Culture media, cell cultures and methods of culturing pluripotent stem cells in an undifferentiated state

ABSTRACT

Provided are novel serum-free culture media which comprise basic fibroblast growth factor (bFGF), transforming growth factor beta-3 and ascorbic acid at a concentration of at least about 50 microgram/ml; ascorbic acid at a concentration range of about 400-600 microgram/ml, bFGF at a concentration range of about 50-200 ng/ml, xeno-free serum replacement and a lipid mixture; the IL6RIL6 chimera at a concentration range of about 50-200 picogram per milliliter (pg/ml); or leukemia inhibitory factor (LIF) at a concentration of at least 2000 units/ml; cell cultures comprising same with pluripotent stem cells such as human embryonic stem cells and induced pluripotent stem (iPS) cells, and methods of using same for expanding pluripotent stem cells in an undifferentiated state using two-dimensional or three-dimensional culture systems; and methods of expanding iPS cells in a suspension culture devoid of substrate adherence and cell encapsulation.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.14/696,520 filed on Apr. 27, 2015, which is a continuation of U.S.patent application Ser. No. 13/508,991 filed on May 10, 2012, now U.S.Pat. No. 9,018,010, which is a National Phase of PCT Patent ApplicationNo. PCT/IL2010/000937 having International Filing Date of Nov. 11, 2010,which claims the benefit of priority of U.S. Provisional PatentApplication No. 61/272,860 filed on Nov. 12, 2009. The contents of theabove applications are all incorporated by reference as if fully setforth herein in their entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 84991SequenceListing.txt, created on Nov. 11,2020, comprising 35,204 bytes, submitted concurrently with the filing ofthis application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to xeno-freeculture media which can be used to maintain stem cells in a pluripotentand undifferentiated state, and in some embodiments to defined culturemedia, cell cultures comprising same and methods using same forculturing pluripotent stem cells in a suspension culture.

The exceptional differentiation potential of human embryonic stem cells(hESCs) underlines them as one of the best models to study early humandevelopment, lineage commitment, differentiation processes and to beused for industrial purposes and cell-based therapy.

Induced pluripotent (iPS) cells are somatic cells which arere-programmed to ESC-like cells capable of differentiation intorepresentative tissues of the three embryonic germ layers both in vitroand in vivo. Mouse or human iPS cells were generated by over expressionof four transcription factors, c-Myc, Oct4, Klf4 and Sox2 in somaticcells. The iPS cells were shown to form the same colony morphology asESCs and to express some typical ESCs markers such as Myb, Kit, Gdf3 andZic3, but less prominently markers such as Dnmt3a, Dnmt3b, Utf1, Tcl1and the LIF receptor gene, confirming that iPS cells are similar but notidentical to ES cells [Takahashi and Yamanaka, 2006; Takahashi et al,2007; Meissner et al. 2007; Okita et al, 2007]. Yu Junying et al.(Science 318:1917-1920, 2007) found a common gene expression pattern tofibroblasts-derived iPS cells and hESCs.

Further studies revealed that iPS cells could be obtained bytransforming somatic cells with Oct4, Sox2. Nanog and Lin28 whileomitting the use of the oncogene C-Myc [Yu et al, 2007; Nakagawa et al.2008]. Improvements of iPS cells derivation methods include the use ofplasmids instead of viral vectors or derivation without any integrationto the genome, which might simplify the future use of iPS cells forclinical applications [Yu J, et al., Science. 2009, 324: 797-801].

The currently available iPS cells are those derived from embryonicfibroblasts [Takahashi and Yamanaka, 2006; Meissner et al, 2007],fibroblasts formed from hESCs [Park et al. 2008]. Fetal fibroblasts [Yuet al, 2007; Park et al, 2008], foreskin fibroblast [Yu et al, 2007;Park et al, 2008], adult dermal and skin tissues [Hanna et al. 2007;Lowry et al. 2008], b-lymphocytes [Hanna et al 2007] and adult liver andstomach cells [Aoi et al. 2008].

Similarly to hESCs, iPS cells are traditionally cultured with asupportive layer in 2D culture, which allows their continuous growth inthe undifferentiated state. For example, iPS cells were cultured onfeeder-layers consisting of inactivated mouse embryonic fibroblasts(MEF) or foreskin fibroblasts [Takahashi and Yamanaka 2006, Meisnner atal 2007] in the presence of a medium supplemented with fetal bovineserum (FBS). Further improvements of the culturing methods includeculturing iPS cells on MEF feeder layers in the presence of a moredefined culture medium containing serum replacement and 10 ng/ml ofbasic fibroblasts growth factor (bFGF) (Park et al., 2008). However, forclinical applications (e.g., cell-based therapy) or industrial purposes,the iPS cells should be cultured in a defined, xeno-free (e.g.,animal-free) and a scalable culture system with controlled processes.

PCT Publication No. WO2007/026353 discloses a well-defined, xeno-freeculture media which comprise a TGF-beta isoform or the chimera formedbetween IL6 and the soluble IL6 receptor (IL6RIL6) for maintaining humanembryonic stem cells, in an undifferentiated state in a two-dimensionalculture system.

U.S. Patent Application No. 20050233446 discloses a defined medium whichcomprises bFGF, insulin and ascorbic acid for maintaining hESCs whencultured on Matrigel™ in an undifferentiated state.

Ludwig T E., et al., 2006 (Nature Biotechnology, 24: 185-7) disclosesthe TeSR1 defined medium for culturing hESCs on a matrix composed ofCollagen IV, fibronectin, laminin and virtonectin.

U.S. Patent Application No. 20090029462 discloses methods of expandingpluripotent stem cells in suspension using microcarriers or cellencapsulation.

PCT Publication No. WO/2008/015682 discloses a method of expanding andmaintaining human embryonic stem cells in a suspension culture underculturing conditions devoid of substrate adherence.

U.S. Patent Application No. 20070155013 discloses a method of growingpluripotent stem cells in suspension using a carrier which adheres tothe pluripotent stem cells.

U.S. Patent Application No. 20080241919 (Parsons et al.) discloses amethod of culturing pluripotent stem cells in a suspension culture in amedium which comprises bFGF, insulin and ascorbic acid in a cell culturevessel that includes a cell-free matrix.

U.S. Patent Application No. 20080159994 (Mantalaris et al.) discloses amethod of culturing pluripotent ES cells encapsulated within alginatebeads in a three-dimensional culture in a medium which comprises serumreplacement and bFGF.

U.S. Patent Application No. 20070264713 discloses a method of culturingundifferentiated stem cells in suspension on microcarriers in vesselsusing a conditioned medium.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a culture medium being serum-free and xeno-freecomprising basic fibroblast growth factor (bFGF), transforming growthfactor beta-3 (TGFβ3) and ascorbic acid, wherein a concentration of theascorbic acid in the culture medium is at least about 50 μg/ml andwherein the culture medium is capable of maintaining pluripotent stemcells in an undifferentiated state in the absence of feeder cellsupport.

According to an aspect of some embodiments of the present inventionthere is provided a culture medium being serum-free and xeno-freecomprising ascorbic acid at a concentration range of about 400-600μg/ml, basic fibroblast growth factor (bFGF) at a concentration range ofabout 50-200 ng/ml, xeno-free serum replacement and a lipid mixture,wherein the culture medium is capable of maintaining pluripotent stemcells in an undifferentiated state in the absence of feeder cellsupport.

According to an aspect of some embodiments of the present inventionthere is provided a culture medium being serum-free comprising anIL6RIL6 chimera at a concentration range of about 50-200 picogram permilliliter (pg/ml), wherein the culture medium is capable of maintainingpluripotent stem cells in an undifferentiated state in the absence offeeder cell support.

According to an aspect of some embodiments of the present inventionthere is provided a culture medium being serum-free comprising aleukemia inhibitory factor (LIF) at a concentration of at least 2000units/ml, wherein the culture medium is capable of maintainingpluripotent stem cells in an undifferentiated state in the absence offeeder cell support.

According to an aspect of some embodiments of the present inventionthere is provided a culture medium comprising basic fibroblast growthfactor (bFGF) at a concentration range of about 50-200 ng/ml and serumreplacement, wherein the culture medium is capable of maintainingpluripotent stem cells in an undifferentiated state in a suspensionculture.

According to an aspect of some embodiments of the present inventionthere is provided a culture medium consisting of a basic medium,ascorbic acid at a concentration range of about 50 μg/ml to about 500μg/ml, bFGF at a concentration range between about 2 ng/ml to about 20ng/ml, L-glutamine, and serum replacement.

According to an aspect of some embodiments of the present inventionthere is provided a culture medium consisting of a basic medium,ascorbic acid at a concentration range of about 50 μg/ml to about 500μg/ml, bFGF at a concentration range between about 2 ng/ml to about 20ng/ml, L-glutamine, serum replacement and a lipid mixture.

According to an aspect of some embodiments of the present inventionthere is provided a cell culture comprising a pluripotent stem cell andthe culture medium of the invention.

According to an aspect of some embodiments of the present inventionthere is provided a method of deriving an embryonic stem cell line,comprising: (a) obtaining an embryonic stem cell from a pre-implantationstage blastocyst, post-implantation stage blastocyst and/or a genitaltissue of a fetus; and (b) culturing the embryonic stem cell in theculture medium of the invention; thereby deriving the embryonic stemcell line.

According to an aspect of some embodiments of the present inventionthere is provided a method of deriving an induced pluripotent stem cellline, comprising: (a) inducing a somatic cell to a pluripotent stemcell; and (b) culturing the pluripotent stem cell in the culture mediumof the invention; thereby deriving the induced pluripotent stem cellline.

According to an aspect of some embodiments of the present inventionthere is provided a method of expanding and maintaining pluripotent stemcells in an undifferentiated state, the method comprising culturing thepluripotent stem cells in the culture medium of the invention, therebyexpanding and maintaining the pluripotent stem cells in theundifferentiated state.

According to an aspect of some embodiments of the present inventionthere is provided a method of expanding and maintaining pluripotent stemcells in an undifferentiated state, the method comprising culturing thepluripotent stem cells in a culture medium being serum-free,feeder-free, matrix-free and protein carrier-free and comprising basicfibroblast growth factor (bFGF) at a concentration range of about 50-200ng/ml, wherein the culture medium is capable of maintaining pluripotentstem cells in an undifferentiated state.

According to an aspect of some embodiments of the present inventionthere is provided a method of expanding pluripotent stem cells andmaintaining the pluripotent stem cells in an undifferentiated state, themethod comprising culturing the pluripotent stem cells on a feeder celllayer in a serum-free and xeno-free culture medium, the culture mediumcomprises basic fibroblast growth factor (bFGF) transforming growthfactor beta-3 (TGFβ3) and ascorbic acid, wherein a concentration of theascorbic acid in the culture medium is at least 50 μg/ml and wherein theculture medium is capable of maintaining pluripotent stem cells in anundifferentiated state, thereby expanding and maintaining the stem cellsin the undifferentiated state.

According to an aspect of some embodiments of the present inventionthere is provided a method of expanding pluripotent stem cells andmaintaining the pluripotent stem cells in an undifferentiated state, themethod comprising culturing the pluripotent stem cells on a feeder celllayer in a serum-free and xeno-free culture medium, the culture mediumcomprises ascorbic acid at a concentration range of about 400-600 μg/ml,basic fibroblast growth factor (bFGF) at a concentration range of about50-200 ng/ml, xeno-free serum replacement and a lipid mixture, whereinthe culture medium is capable of maintaining pluripotent stem cells inan undifferentiated state, thereby expanding and maintaining the stemcells in the undifferentiated state.

According to an aspect of some embodiments of the present inventionthere is provided a method of expanding induced pluripotent stem (iPS)cells and maintaining the iPS cells in an undifferentiated state, themethod comprising culturing the iPS cells in a suspension culture underculturing conditions devoid of substrate adherence and devoid of cellencapsulation and which allow expansion of the iPS cells in theundifferentiated state, thereby expanding and maintaining the iPS cellsin the undifferentiated state.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating lineage-specific cells frompluripotent stem cells, the method comprising: (a) culturing thepluripotent stem cells according to the method of the invention, tothereby obtain expanded, undifferentiated stem cells; (b) subjecting theexpanded, undifferentiated stem cells to culturing conditions suitablefor differentiating and/or expanding lineage specific cells; therebygenerating the lineage-specific cells from the pluripotent stem cells.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating embryoid bodies frompluripotent stem cells, the method comprising: (a) culturing thepluripotent stem cells according to the method of the invention, tothereby obtain expanded, undifferentiated pluripotent stem cells; and(b) subjecting the expanded, undifferentiated pluripotent stem cells toculturing conditions suitable for differentiating the stem cells toembryoid bodies; thereby generating the embryoid bodies from thepluripotent stem cells.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating lineage-specific cells frompluripotent stem cells, the method comprising: (a) culturing thepluripotent stem cells according to the method of the invention, tothereby obtain expanded, undifferentiated pluripotent stem cells; (b)subjecting the expanded, undifferentiated pluripotent stem cells toculturing conditions suitable for differentiating the expanded,undifferentiated stem cells to embryoid bodies; and (c) subjecting cellsof the embryoid bodies to culturing conditions suitable fordifferentiating and/or expanding lineage specific cells; therebygenerating the lineage-specific cells from the pluripotent stem cells.

According to some embodiments of the invention, the cell culture isfeeder cells free.

According to some embodiments of the invention, the culture medium iscapable of expanding the pluripotent stem cells in an undifferentiatedstate when cultured in a suspension culture.

According to some embodiments of the invention, the stem cells areembryonic stem cells.

According to some embodiments of the invention, the stem cells areinduced pluripotent stem (iPS) cells.

According to some embodiments of the invention, the embryonic stem cellsare human embryonic stem cells.

According to some embodiments of the invention, the induced pluripotentstem cells are human induced pluripotent stem cells.

According to some embodiments of the invention, the culture medium iscapable of expanding the pluripotent stem cells in an undifferentiatedstate.

According to some embodiments of the invention, the culture mediumfurther comprises basic fibroblast growth factor (bFGF).

According to some embodiments of the invention, the culture mediumfurther comprises serum replacement.

According to some embodiments of the invention, a concentration of theTGFβ3 in the culture medium is at least about 0.5 ng/ml.

According to some embodiments of the invention, a concentration of theTGFβ3 in the culture medium is about 2 ng/ml.

According to some embodiments of the invention, a concentration of thebFGF in the culture medium is at least about 5 ng/ml.

According to some embodiments of the invention, a concentration of thebFGF in the culture medium is in the range of about 5 ng/ml to about 200ng/ml.

According to some embodiments of the invention, a concentration of theascorbic acid in the culture medium is in the range of about 400microgram/milliliter (μg/ml) to about 600 μg/ml.

According to some embodiments of the invention, a concentration of theascorbic acid in the culture medium is about 500 μg/m(microgram/milliliter).

According to some embodiments of the invention, the culturing iseffected on a matrix.

According to some embodiments of the invention, the matrix comprises anextracellular matrix.

According to some embodiments of the invention, the extracellular matrixis selected from the group consisting of a fibronectin matrix, a lamininmatrix, and a foreskin fibroblast matrix.

According to some embodiments of the invention, the matrix is xeno-free.

According to some embodiments of the invention, the feeder cell layer isxeno-free.

According to some embodiments of the invention, the feeder cell layercomprises foreskin fibroblast cells.

According to some embodiments of the invention, the bFGF is at aconcentration range of about 0.1 ng/ml to about 500 ng/ml, the TGFβ3 isat a concentration range of about 0.1 ng/ml to about 20 ng/ml, theascorbic acid is at a concentration range of about 50 μg/ml to about5000 μg/ml.

According to some embodiments of the invention, the bFGF is at aconcentration range of about 5 ng/ml to about 150 ng/ml, the TGFβ3 is ata concentration range of about 0.5 ng/ml to about 5 ng/ml, the ascorbicacid is at a concentration range of about 400 μg/ml to about 600 g/ml.

According to some embodiments of the invention, the culture mediumfurther comprising serum replacement.

According to some embodiments of the invention, the serum replacement isxeno free.

According to some embodiments of the invention, the culture mediumfurther comprising a lipid mixture.

According to some embodiments of the invention, the culture mediumfurther comprising sodium bicarbonate at a concentration of about 5% toabout 10%.

According to some embodiments of the invention, the lipid mixture is ata concentration of about 1%.

According to some embodiments of the invention, the concentration of theIL6RIL6 chimera is about 100 μg/ml.

According to some embodiments of the invention, the concentration of theLIF is in a range of about 2000-4000 units/ml.

According to some embodiments of the invention, the culturing iseffected in a suspension culture.

According to some embodiments of the invention, the culture medium isdevoid of TGFβ3.

According to some embodiments of the invention, the culture mediumcomprises no more than 0.1 ng/ml of TGFβ3.

According to some embodiments of the invention, a culture medium of thesuspension culture is serum-free and feeder cell-free.

According to some embodiments of the invention, the culture medium beingserum-free and devoid of animal contaminants.

According to some embodiments of the invention, the concentration ofsaid bFGF is about 1 ng/ml.

According to some embodiments of the invention, the culture mediumcomprises an IL6RIL6 chimera at a concentration range of about 50-200picograms per milliliter (pg/ml), wherein the culture medium is capableof maintaining the iPS cells in an undifferentiated state in the absenceof feeder cell support.

According to some embodiments of the invention, the culture mediumcomprises leukemia inhibitory factor (LIF) at a concentration of atleast 2000 units/ml wherein the culture medium is capable of maintainingthe iPS cells in an undifferentiated state in the absence of feeder cellsupport.

According to some embodiments of the invention, the culture mediumcomprises basic fibroblast growth factor (bFGF) at a concentration rangeof about 50-200 ng/ml.

According to some embodiments of the invention, the culture mediumcomprises an IL6RIL6 chimera at a concentration range of about 50-200nanogram per milliliter (ng/ml).

According to some embodiments of the invention, the culture mediumfurther comprises basic fibroblast growth factor (bFGF).

According to some embodiments of the invention, the culture medium isprotein carrier-free.

According to some embodiments of the invention, expanding comprisesobtaining at least about 8×10⁶ cells from a single pluripotent stem cellfollowing about 1 month.

According to some embodiments of the invention, the pluripotent stemcells cultured in the culture medium exhibits a normal chromosomalkaryotype following at least 2 passages.

According to some embodiments of the invention, the pluripotent stemcells exhibits a doubling time of at least 20 hours.

According to some embodiments of the invention, maintaining is for atleast 5 passages.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-1C are photographs depicting colony morphology of iPS cellscultured on a xeno-free two-dimensional culture system in the presenceof the novel xeno-free (e.g., animal-free, devoid of animalcontamination) culture media according to some embodiments of theinvention. J1.2-3 were cultured with human foreskin fibroblast (HFF)supportive layers while using the following animal-serum free culturemedium: FIG. 1A—medium HA70 for 6 passages; FIG. 1B—medium HA40/4 for 6passages; and FIG. 1C—medium D2 for 16 passages.

FIGS. 2A-2C are photographs depicting immuno-fluorescence staining ofiPS cells with markers of pluripotency. J1.2-3 and iF4 iPS cells werecultured on a xeno-free two-dimensional culture system (HFF) in thepresence of the animal-serum free culture medium HA77 for at least 10passages and were then stained with the following markers ofundifferentiated markers: FIG. 2A—J1.2-3 iPS cells stained with Oct4;FIG. 2B—iF4 iPS cells stained with SSEA4; and FIG. 2C—iF4 iPS cellsstained with TRA-1-81.

FIGS. 3A-3C are photographs depicting the morphology of the J1.2-3 iPScell line from HFF when cultured in suspension in the followingxeno-free culture media for the indicated passages. FIG. 3A—J1.2-3 iPScells cultured in the yFL3 medium for 16 passages; FIG. 3B—J1.2-3 iPScells cultured in the CM100F medium for 13 passages; FIG. 3C—J1.2-3 iPScells cultured in the yF100 medium for 8 passages. Note that whilecultured in suspension the iPS cells create sphere like structurecontaining undifferentiated cells.

FIG. 4 is a photograph depicting the morphology of the J1.2-3 iPS cellswhen cultured on mouse embryonic fibroblasts (MEF) after an extendedculturing period in a suspension culture. J1.2-3 cells were cultured for37 passages in suspension in the CM100F medium, following which theywere re-cultured with MEFs. and form typical iPS colony morphology 24hours post their culture with MEFs.

FIGS. 5A-5C are photographs depicting immuno-fluorescence staining ofiPS cells with markers of pluripotency. J1.2-3 cells were cultured insuspension using medium CM100F for more than 20 passages and were thenstained with markers of undifferentiated stem cells. FIG. 5A—TRA-1-81;FIG. 5B—TRA-1-60; FIG. 5C—SSEA4.

FIGS. 6A-6D are photographs depicting immunostaining of iPS cells withmarkers of pluripotency. J1.2-3 cells were cultured in suspension usingthe CM100F medium for at least 30 passages and then were transferredinto spinner flasks and were cultured for additional 30 days, followingwhich the cells were stained with markers of undifferentiated stemcells. FIG. 6A—Oct4; FIG. 6B—TRA-1-81; FIG. 6C—TRA-1-60; and FIG.6D—SSEA4.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to novelculture media, cell cultures comprising same and methods utilizing samefor maintaining pluripotent stem cells in a proliferative, pluripotentand undifferentiated state and, more particularly, but not exclusively,to methods of expanding hESCs and induced pluripotent stem (iPS) cellsin suspension cultures or two-dimensional culture systems whilemaintaining the cells in a proliferative, pluripotent andundifferentiated state.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

The present inventors have designed following laborious experimentationsdefined culture media, which are serum-free and xeno-free (e.g., devoidof animal contaminants) and which can maintain pluripotent stem cellssuch as human iPS and ESCs in an undifferentiated state in the absenceof feeder cell support while preserving their pluripotent potential todifferentiae into all three embryonic germ layers.

Thus, as shown in the Examples section which follows, hESCs and iPScells were cultured in an undifferentiated state on two-dimensionalculture systems which are either feeder-layer free (e.g., on a syntheticmatrix; Example 1) or xeno-free feeder layer-based (e.g., foreskinfibroblasts; FIGS. 1A-C and 2A-C, Example 2) in the presence ofserum-free, xeno-free and defined culture media (e.g., mHA40/4. HA75,HA76, HA77, HA78 or HA74). While in culture, the pluripotent stem cellsexhibit undifferentiated morphology, as well as morphological andmolecular characteristics typical to iPS or hESCs such as normalkaryotype, expression of markers of pluripotency (e.g., Oct4, SSEA4,TRA-1-81. TRA-1-60), and ability to differentiate into all threeembryonic germ layers both in vitro (by formation of embryoid bodiesafter at least 28 passages) and in vivo (by formation of teratomas afterat least 31 passages).

As used herein the phrase “pluripotent stem cells” refers to cells whichare capable of differentiating into cells of all three embryonic germlayers (i.e., endoderm, ectoderm and mesoderm). According to someembodiments of the invention, the phrase “pluripotent stem cells”encompasses embryonic stem cells (ESCs) and induced pluripotent stemcells (iPS cells).

The phrase “embryonic stem cells” may comprise cells which are obtainedfrom the embryonic tissue formed after gestation (e.g., blastocyst)before implantation (i.e., a pre-implantation blastocyst), extendedblastocyst cells (EBCs) which are obtained from apost-implantation/pre-gastrulation stage blastocyst (see WO2006/040763]and embryonic germ (EG) cells which are obtained from the genital tissueof a fetus any time during gestation, preferably before 10 weeks ofgestation.

According to some embodiments of the invention, the pluripotent stemcells of the invention are embryonic stem cells, such as from a human orprimate (e.g., monkey) origin.

The embryonic stem cells of the invention can be obtained usingwell-known cell-culture methods. For example, human embryonic stem cellscan be isolated from human blastocysts. Human blastocysts are typicallyobtained from human in vivo preimplantation embryos or from in vitrofertilized (IVF) embryos. Alternatively, a single cell human embryo canbe expanded to the blastocyst stage. For the isolation of human ES cellsthe zona pellucida is removed from the blastocyst and the inner cellmass (ICM) is isolated by immunosurgery, in which the trophectodermcells are lysed and removed from the intact ICM by gentle pipetting. TheICM is then plated in a tissue culture flask containing the appropriatemedium which enables its outgrowth. Following 9 to 15 days, the ICMderived outgrowth is dissociated into clumps either by a mechanicaldissociation or by an enzymatic degradation and the cells are thenre-plated on a fresh tissue culture medium. Colonies demonstratingundifferentiated morphology are individually selected by micropipette,mechanically dissociated into clumps, and re-plated. Resulting ES cellsare then routinely split every 4-7 days. For further details on methodsof preparation human ES cells see Thomson et al., [U.S. Pat. No.5,843,780; Science 282: 1145, 1998; Curr. Top. Dev. Biol. 38: 133, 1998;Proc. Natl. Acad. Sci. USA 92: 7844, 1995]; Bongso et al., [Hum Reprod4: 706, 1989]; and Gardner et al., [Fertil. Steril. 69: 84, 1998].

It will be appreciated that commercially available stem cells can alsobe used with this aspect of the present invention. Human ES cells can bepurchased from the NIH human embryonic stem cells registry(wwwdotescrdotnihdotgov). Non-limiting examples of commerciallyavailable embryonic stem cell lines are BG01, BG02, BG03, BG04, CY12,CY30, CY92, CY10, TE03, TE04 and TE06.

Extended blastocyst cells (EBCs) can be obtained from a blastocyst of atleast nine days post fertilization at a stage prior to gastrulation.Prior to culturing the blastocyst, the zona pellucida is digested [forexample by Tyrode's acidic solution (Sigma Aldrich, St Louis. Mo., USA)]so as to expose the inner cell mass. The blastocysts are then culturedas whole embryos for at least nine and no more than fourteen days postfertilization (i.e., prior to the gastrulation event) in vitro usingstandard embryonic stem cell culturing methods.

Embryonic germ (EG) cells are prepared from the primordial germ cellsobtained from fetuses of about 8-11 weeks of gestation (in the case of ahuman fetus) using laboratory techniques known to anyone skilled in thearts. The genital ridges are dissociated and cut into small chunks whichare thereafter disaggregated into cells by mechanical dissociation. TheEG cells are then grown in tissue culture flasks with the appropriatemedium. The cells are cultured with daily replacement of medium until acell morphology consistent with EG cells is observed, typically after7-30 days or 1-4 passages. For additional details on methods ofpreparation human EG cells see Shamblott et al., [Proc. Natl. Acad. Sci.USA 95: 13726, 1998] and U.S. Pat. No. 6,090,622.

The phrase “induced pluripotent stem (iPS) cell” (or embryonic-like stemcell) as used herein refers to a proliferative and pluripotent stem cellwhich is obtained by de-differentiation of a somatic cell (e.g., anadult somatic cell).

According to some embodiments of the invention, the iPS cell ischaracterized by a proliferative capacity which is similar to that ofESCs and thus can be maintained and expanded in culture for an almostunlimited time.

IPS cells can be endowed with pluripotency by genetic manipulation whichre-program the cell to acquire embryonic stem cells characteristics. Forexample, the iPS cells of the invention can be generated from somaticcells by induction of expression of Oct-4, Sox2, Kfl4 and c-Myc in asomatic cell essentially as described in Takahashi and Yamanaka, 2006,Takahashi et al, 2007, Meissner et al. 2007, and Okita et al, 2007).Additionally or alternatively, the iPS cells of the invention can begenerated from somatic cells by induction of expression of Oct4. Sox2,Nanog and Lin28 essentially as described in Yu et al, 2007, and Nakagawaet al, 2008. It should be noted that the genetic manipulation(re-programming) of the somatic cells can be performed using any knownmethod such as using plasmids or viral vectors, or by derivation withoutany integration to the genome [Yu J. et al., Science. 2009, 324:797-801].

The iPS cells of the invention can be obtained by inducingde-differentiation of embryonic fibroblasts [Takahashi and Yamanaka.2006: Meissner et al, 2007], fibroblasts formed from hESCs [Park et al,2008], Fetal fibroblasts [Yu et al. 2007; Park et al, 2008], foreskinfibroblast [Yu et al. 2007; Park et al, 2008], adult dermal and skintissues [Hanna et al. 2007; Lowry et al. 2008], b-lymphocytes [Hanna etal 2007] and adult liver and stomach cells [Aoi et al. 2008].

IPS cell lines are also available via cell banks such as the WiCellbank. Non-limiting examples of commercially available iPS cell linesinclude the iPS foreskin clone 1 [WiCell Catalogue No.iPS(foreskin)-1-DL-1], the iPSIMR90 clone 1 [WiCell Catalogue No.iPS(IMR90)-1-DL-1], and the iPSIMR90 clone 4 [WiCell Catalogue No.iPS(IMR90)-4-DL-1].

According to some embodiments of the invention, the induced pluripotentstem cells are human induced pluripotent stem cells.

As used herein the phrase “culture medium” refers to a liquid substanceused to support the growth of pluripotent stem cells and maintain themin an undifferentiated state. The culture medium used by the inventionaccording to some embodiments can be a water-based medium which includesa combination of substances such as salts, nutrients, minerals,vitamins, amino acids, nucleic acids, proteins such as cytokines, growthfactors and hormones, all of which are needed for cell proliferation andare capable of maintaining the pluripotent stem cells in anundifferentiated state. For example, a culture medium according to anaspect of some embodiments of the invention can be a synthetic tissueculture medium such as the Ko-DMEM (Gibco-Invitrogen Corporationproducts, Grand Island, N.Y., USA), DMEM/F12 (Biological Industries,Biet HaEmek, Israel), Mab ADCB medium (HyClone, Utah, USA) supplementedwith the necessary additives as is further described hereinunder.

The phrase “feeder cell support” as used herein refers to the ability ofa feeder cell (e.g., fibroblasts) to maintain pluripotent stem cells ina proliferative and undifferentiated state when the pluripotent stemcells are co-cultured on the feeder cells or when the pluripotent stemcells are cultured on a matrix (e.g., an extracellular matrix, asynthetic matrix) in the presence of a conditioned medium generated bythe feeder cells. The support of the feeder cells depends on thestructure of the feeder cells while in culture (e.g., the threedimensional matrix formed by culturing the feeder cells in a tissueculture plate), function of the feeder cells (e.g., the secretion ofgrowth factors, nutrients and hormones by the feeder cells, the growthrate of the feeder cells, the expansion ability of the feeder cellsbefore senescence) and/or the attachment of the pluripotent stem cellsto the feeder cell layer(s).

The phrase “absence of feeder cell support” as used herein refers to aculture medium and/or a cell culture being devoid of feeder cells and/ora conditioned medium generated thereby.

As used herein the phrase “serum-free” refers to being devoid of a humanor an animal serum.

It should be noted that the function of serum in culturing protocols isto provide the cultured cells with an environment similar to thatpresent in vivo (i.e., within the organism from which the cells arederived, e.g., a blastocyst of an embryo). However, the use of serum,which is derived from either an animal source (e.g., bovine serum) or ahuman source (human serum), is limited by the significant variations inserum components between the donor individuals (from which the serum isobtained) and the risk of having xeno contaminants (in case of an animalserum is used).

According to some embodiments of the invention, the serum-free culturemedium does not comprise serum or portions thereof.

According to some embodiments of the invention, the serum-free culturemedium of the invention is devoid of serum albumin (e.g., albumin whichis purified from human serum or animal serum).

According to some embodiments of the invention the culture mediumcomprises serum replacement.

As used herein the phrase “serum replacement” refers to a definedformulation, which substitutes the function of serum by providingpluripotent stem cells with components needed for growth and viability.

Various serum replacement formulations are known in the art and arecommercially available.

For example, GIBCO™ KNOCKOUT™ Serum Replacement (Gibco-InvitrogenCorporation. Grand Island, N.Y. USA, Catalogue No. 10828028) is adefined serum-free formulation optimized to grow and maintainundifferentiated ES cells in culture. It should be noted that theformulation of GIBCO™ Knockout™ Serum Replacement includes Albumax(Bovine serum albumin enriched with lipids) which is from an animalsource (International Patent Publication No. WO 98/30679 to Price, P. J.et al). However, a recent publication by Crook et al., 2007 (Crook J M.,et al., 2007. Cell Stem Cell, 1: 490-494) describes six clinical-gradehESC lines generated using FDA-approved clinical grade foreskinfibroblasts in cGMP-manufactured KNOCKOUT™ Serum Replacement (InvitrogenCorporation. USA, Catalogue No. 04-0095).

According to some embodiments of the invention, the concentration ofGIBCO™ Knockout KNOCKOUT™ Serum Replacement in the culture medium is inthe range of from about 1% [volume/volume (v/v)] to about 50% (v/v),e.g., from about 5% (v/v) to about 40% (v/v), e.g., from about 5% (v/v)to about 30% (v/v), e.g., from about 10% (v/v) to about 30% (v/v), e.g.,from about 10% (v/v) to about 25% (v/v), e.g., from about 10% (v/v) toabout 20% (v/v), e.g., about 10% (v/v). e.g., about 15% (v/v). e.g.,about 20% (v/v), e.g., about 30% (v/v).

Another commercially available serum replacement is the B27 supplementwithout vitamin A which is available from Gibco-Invitrogen, Corporation.Grand Island, N.Y. USA. Catalogue No. 12587-010. The B27 supplement is aserum-free formulation which includes d-biotin, fatty acid free fractionV bovine serum albumin (BSA), catalase, L-carnitine HCl, corticosterone,ethanolamine HCl, D-galactose (Anhyd.), glutathione (reduced),recombinant human insulin, linoleic acid, linolenic acid, progesterone,putrescine-2-HCl, sodium selenite, superoxide dismutase, T-3/albumincomplex. DL alpha-tocopherol and DL alpha tocopherol acetate. However,the use of B27 supplement is limited since it includes albumin from ananimal source.

According to some embodiments of the invention, the serum replacement isxeno-free.

The term “xeno” is a prefix based on the Greek word “Xenos”, i.e., astranger. As used herein the phrase “xeno-free” refers to being devoidof any components which are derived from a xenos (i.e., not the same, aforeigner) species. Such components can be contaminants such aspathogens associated with (e.g., infecting) the xeno species, cellularcomponents of the xeno species or a-cellular components (e.g., fluid) ofthe xeno species.

For example, a xeno-free serum replacement can include a combination ofinsulin, transferrin and selenium. Additionally or alternatively, axeno-free serum replacement can include human or recombinantly producedalbumin, transferrin and insulin.

Non-limiting examples of commercially available xeno-free serumreplacement compositions include the premix of ITS (Insulin. Transferrinand Selenium) available from Invitrogen corporation (ITS, Invitrogen,Catalogue No. 5150-056); Serum replacement 3 (Sigma. Catalogue No.S2640) which includes human serum albumin, human transferring and humanrecombinant insulin and does not contain growth factors, steroidhormones, glucocorticoids, cell adhesion factors, detectable Ig andmitogens.

According to some embodiments of the invention, the xeno-free serumreplacement formulations ITS (Invitrogen corporation) and SR3 (Sigma)are diluted in a 1 to 100 ratio in order to reach a ×1 workingconcentration.

According to some embodiments of the invention the culture medium iscapable of maintaining pluripotent stem cell in a proliferative,pluripotent and undifferentiated state for at least about 5 passages, atleast about 10 passages, at least about 15 passages, at least about 20passages, at least about 22 passages, at least about 25 passages, atleast about 30 passages, at least about 35 passages, at least about 40passages, at least about 45 passages, at least about 50 passages andmore.

According to some embodiments of the invention the culture medium iscapable of expanding the pluripotent stem cells in an undifferentiatedstate.

As used herein the term “expanding” refers to increasing the number ofpluripotent stem cells over the culturing period (by at least about 5%,10%, 15%, 20%, 30%, 50%, 100%, 200%, 500%, 1000%, and more). It will beappreciated that the number of pluripotent stem cells which can beobtained from a single pluripotent stem cell depends on theproliferation capacity of the pluripotent stem cell. The proliferationcapacity of a pluripotent stem cell can be calculated by the doublingtime of the cell (i.e., the time needed for a cell to undergo a mitoticdivision in the culture) and the period the pluripotent stem cellculture can be maintained in the undifferentiated state (which isequivalent to the number of passages multiplied by the days between eachpassage).

For example, as described in Example 1 of the Examples section whichfollows, the hESCs or human iPS cells could be maintained in theproliferative, pluripotent and undifferentiated state in the presence ofthe mHA40/4, HA75, HA76, HA78 and HA74/1 culture media for at least 22passages when cultured on a feeder-free matrix. Given that each passageoccurs every 4-7 days, the hESCs or human iPS cells were maintained for110 days (i.e., 2640 hours). Given that the hESCs or human iPS doublingtime was 36 hours, a single hESC or human iPS cell cultured under theseconditions could be expanded to give rise to 2⁷³ (i.e., 9.4×10²¹) hESCsor human iPS cells.

According to some embodiments of the invention, the culture medium ofsome embodiments of the invention is capable of supporting expansion ofa single pluripotent stem cell (e.g., hESC or human iPS cell) or apopulation of pluripotent stem cells by at least 23 (i.e., 8×10⁶) withinabout one month, e.g., at least 2²⁴ (i.e., 16.7×10⁶) within about onemonth.

According to some embodiments of the invention the serum-free andxeno-free culture medium comprises basic fibroblast growth factor(bFGF), transforming growth factor beta-3 (TGFβ₃) and ascorbic acid,wherein a concentration of the ascorbic acid in the culture medium is atleast 50 μg/ml and wherein the culture medium is capable of maintainingpluripotent stem cells in an undifferentiated state in the absence offeeder cell support.

Ascorbic acid (also known as vitamin C) is a sugar acid (C₆H₈O₆;molecular weight 176.12 grams/mole) with antioxidant properties. Theascorbic acid used by the culture medium of some embodiments of theinvention can be a natural ascorbic acid, a synthetic ascorbic acid, anascorbic acid salt (e.g., sodium ascorbate, calcium ascorbate, potassiumascorbate), an ester form of ascorbic acid (e.g., ascorbyl palmitate,ascorbyl stearate), a functional derivative thereof (a molecule derivedfrom ascorbic acid which exhibits the same activity/function when usedin the culture medium of the invention), or an analogue thereof (e.g., afunctional equivalent of ascorbic acid which exhibits an activityanalogous to that observed for ascorbic acid when used in the culturemedium of the invention). Non-limiting examples of ascorbic acidformulations which can be used in the culture medium of some embodimentsof the invention include L-ascorbic acid and ascorbic acid 3-phosphate.

Ascorbic acid can be obtained from various manufacturers such as Sigma,St Louis, Mo., USA (e.g., Catalogue numbers: A2218, A5960, A7506, A0278,A4403, A4544, A2174, A2343, 95209, 33034, 05878, 95210, 95212, 47863,01-6730, 01-6739, 255564, A92902, W210901).

As mentioned, the concentration of ascorbic acid in the culture mediumis at least about 50 μg/ml. According to some embodiments of theinvention, the ascorbic acid can be used in a range of concentrationssuch as from about 50 μg/ml to about 50 mg/ml. e.g., from about 50 μg/mlto about 5 mg/ml. e.g., from about 50 μg/ml to about 1 mg/ml, e.g., fromabout 100 μg/ml to about 800 μg/ml, e.g., from about 200 μg/ml to about800 μg/ml, e.g., from about 300 μg/ml to about 700 μg/ml, e.g., fromabout 400 μg/ml to about 600 μg/ml, e.g., from about 450 μg/ml to about550 μg/ml.

According to some embodiments of the invention the concentration ofascorbic acid in the culture medium is at least about 75 μg/ml, e.g., atleast about 100 μg/ml, e.g., at least about 150 μg/ml, e.g., at leastabout 200 μg/ml, e.g., at least about 250 μg/ml, e.g., at least about300 μg/ml, e.g., at least about 350 μg/ml, e.g., at least about 400μg/ml, e.g., at least about 450 μg/ml, e.g., about 500 μg/ml.

As is shown in Example 1 of the Examples section which follows, thepresent inventors have used various culture media which include ascorbicacid at a concentration of at least 50 μg/ml (e.g., the mHA40/4, HA75,HA76, HA77, HA78 and HA74/1 culture media) to successfully culture hESCsand iPS cells and maintain them in a proliferative, pluripotent andundifferentiated state for at least 15 passages in the absence of feedercell support.

Basic fibroblast growth factor (also known as bFGF, FGF2 or FGF-β) is amember of the fibroblast growth factor family. The bFGF used in theculture medium of some embodiments of the invention can be a purified, asynthetic or a recominantly expressed bFGF protein [(e.g., human bFGFpolypeptide GenBank Accession No. NP_001997.5 (SEQ ID NO:31); human bFGFpolynucleotide GenBank Accession No. NM_002006.4 (SEQ ID NO:32). Itshould be noted that for the preparation of a xeno-free culture mediumthe bFGF is preferably purified from a human source or is recombinantlyexpressed as is further described hereinbelow, bFGF can be obtained fromvarious commercial sources such as Cell Sciences®, Canton, Mass. USA(e.g., Catalogue number CRF001A and CRF001B). Invitrogen Corporationproducts, Grand Island N.Y. USA (e.g., Catalogue numbers: PHG0261,PHG0263, PHG0266 and PHG0264), ProSpec-Tany TechnoGene Ltd. Rehovot,Israel (e.g., Catalogue number: CYT-218), and Sigma, St Louis, Mo., USA(e.g., catalogue number: F0291).

According to some embodiments the concentration of bFGF in culturemedium is in the range from about 1 ng/ml to about 10 μg/ml, e.g., fromabout 2 ng/ml to about 1 μg/ml, e.g., from about 1 ng/ml to about 500ng/ml. e.g., from about 2 ng/ml to about 500 ng/ml, e.g., from about 5ng/ml to about 250 ng/ml, e.g., from about 5 ng/ml to about 200 ng/ml,e.g., from about 5 ng/ml to about 150 ng/ml. e.g., about 10 ng/ml, e.g.,about 20 ng/ml, e.g., about 30 ng/ml, e.g., about 40 ng/ml, e.g., about50 ng/ml, e.g., about 60 ng/ml, e.g., about 70 ng/ml. e.g., about 80ng/ml, e.g., about 90 ng/ml, e.g., about 100 ng/ml, e.g., about 110ng/ml, e.g., about 120 ng/ml, e.g., about 130 ng/ml, e.g., about 140ng/ml, e.g., about 150 ng/ml.

According to some embodiments of the invention the concentration of bFGFin the culture medium is at least about 1 ng/ml, at least about 2 ng/ml,at least about 3 ng, at least about 4 ng/ml, at least about 5 ng/ml, atleast about 6 ng/ml, at least about 7 ng, at least about 8 ng/ml, atleast about 9 ng/ml, at least about 10 ng/ml, at least about 15 ng/ml,at least about 20 ng/ml, at least about 25 ng/ml, at least about 30ng/ml, at least about 35 ng/ml, at least about 40 ng/ml, at least about45 ng/ml, at least about 50 ng/ml, at least about 55 ng/ml, at leastabout 60 ng/ml, at least about 70 ng/ml, at least about 80 ng/ml, atleast about 90 ng/ml, at least about 95 ng/ml, e.g., about 100 ng/ml.

As is shown in Example 1 of the Examples section which follows, thepresent inventors have used various culture media which include bFGF inthe range of 5-200 ng/ml (e.g., the mHA40/4. HA75 and HA78 culturemedia, which include 10 ng/ml bFGF; the HA76 and HA77 culture mediawhich include 100 ng/ml bFGF; and the HA74/1 culture medium whichincludes 50 ng/ml bFGF) to successfully culture hESCs and iPS cells andmaintain them in a proliferative, pluripotent and undifferentiated statefor at least 15 passages in the absence of feeder cell support.

Transforming growth factor beta-3 (TGFβ₃) is involved in the control ofproliferation, differentiation, and other functions in many cell types,acts in inducing transformation and as a negative autocrine growthfactor. TGFβ3 can be obtained from various commercial sources such asR&D Systems Minneapolis Minn., USA.

According to some embodiments of the invention, the concentration ofTGFβ₃ in the culture medium is in the range of about 0.05 ng/ml to about1 μg/ml, e.g., from 0.1 ng/ml to about 1 μg/ml, e.g., from about ofabout 0.5 ng/ml to about 100 ng/ml.

According to some embodiments of the invention, the concentration ofTGFβ₃ in the culture medium is at least about 0.5 ng/ml, e.g., at leastabout 0.6 ng/ml, e.g., at least about 0.8 ng/ml, e.g., at least about0.9 ng/ml, e.g., at least about 1 ng/ml, e.g., at least about 1.2 ng/ml,e.g., at least about 1.4 ng/ml. e.g., at least about 1.6 ng/ml, e.g., atleast about 1.8 ng/ml, e.g., about 2 ng/ml.

As is shown in Example 1 of the Examples section which follows, thepresent inventors have used various culture media which include TGFβ₃ ata concentration of about 2 ng/ml (e.g., the mHA40/4, HA75, HA76, HA78and HA74/1 culture media) to successfully culture hESCs and iPS cellsand maintain them in a proliferative, pluripotent and undifferentiatedstate for at least 22 passages in the absence of feeder cell support.

According to some embodiments of the invention, the culture mediumcomprises bFGF at a concentration range of about 0.1 ng/ml to about 500ng/ml. TGFβ3 at a concentration range of about 0.1 ng/ml to about 20ng/ml, and ascorbic acid at a concentration range of about 50 μg/ml toabout 5000 μg/ml.

According to some embodiments of the invention, the culture medium ofsome embodiments of the invention comprises bFGF at a concentrationrange of about 5 ng/ml to about 150 ng/ml, TGFβ3 at a concentrationrange of about 0.5 ng/ml to about 5 ng/ml, and ascorbic acid at aconcentration range of about 400 μg/ml to about 600 μg/ml.

According to some embodiments of the invention, the culture mediumfurther comprises a lipid mixture.

As used herein the phrase “lipid mixture” refers to a defined (e.g.,chemically defined) lipid composition needed for culturing thepluripotent stem cells. It should be noted that the lipid mixture isusually added to a culture medium which is devoid of serum or serumreplacement and thus substitutes the lipids which are usually added toformulations of serum or serum replacement.

A non-limiting example of a commercially available lipid mixture, whichcan be used in the culture medium of some embodiments of the invention,include the Chemically Define Lipid Concentrate available fromInvitrogen (Catalogue No. 11905-031).

According to some embodiments of the invention, the concentration of thelipid mixture in the culture medium is from about 0.5% [volume/volume(v/v)] to about 3% v/v. e.g., from about 0.5% v/v to about 2% v/v, e.g.,from about 0.5% v/v to about 1% v/v, e.g., about 1% v/v.

According to some embodiments of the invention, the culture medium ofsome embodiments of the invention comprises bFGF at a concentrationrange of about 0.1 ng/ml to about 500 ng/ml, TGFβ₃ at a concentrationrange of about 0.1 ng/ml to about 20 ng/ml, ascorbic acid at aconcentration range of about 50 μg/ml to about 5000 μg/ml, xeno-freeserum replacement and a lipid mixture.

Non-limiting examples of xeno-free and serum-free culture media whichcomprise TGFβ₃, bFGF and ascorbic acid at a concentration of at least 50μg/ml and which can be used to maintain pluripotent stem cells in aproliferative and undifferentiated states include the HA75 and HA78culture media.

According to some embodiments of the invention, the culture mediumfurther comprises sodium bicarbonate. Sodium bicarbonate can be obtainedfrom Biological Industries, Beit HaEmek, Israel.

According to some embodiments of the invention, the concentration ofsodium bicarbonate in the culture medium is from about 5% to about 10%,e.g., from about 6% to about 9%, e.g., from about 7% to about 8%, e.g.,about 7.5%.

The present inventors uncovered that pluripotent stem cells can bemaintained in a proliferative, pluripotent and undifferentiated statefor at least 15 passages when cultured in a serum-free and xeno-freeculture medium which comprises bFGF and ascorbic acid but does notcomprise a TGFβ isoform.

As used herein the phrase “TGFβ isoform” refers to any isoform of thetransforming growth factor beta (0) including TGFβ₁ (e.g., Homo sapiensTGFβ₁. GenBank Accession No. NP_000651), TGFβ₂ (e.g., Homo sapiensTGFβ₂, GenBank Accession No. NP_003229) and TGFβ3 (e.g., Homo sapiensTGFβ₃. GenBank Accession No. NP_003230) which functions through the samereceptor signaling system in the control of proliferation,differentiation, and other functions in many cell types. TGFβ acts ininducing transformation and also acts as a negative autocrine growthfactor.

According to some embodiments of the invention, the culture mediumcomprises no more than 1 ng/ml of the TGFβ isoform, e.g., no more than0.5 ng/ml. e.g., no more than 0.1 ng/ml, e.g., no more than 0.05 ng/ml.e.g., no more than 0.01 ng/ml of the TGFβ isoform.

According to some embodiments of the invention, the culture medium iscompletely devoid of a TGFβ isoform (i.e., TGFβ isoform-free).

According to some embodiments of the invention the culture mediumcomprises ascorbic acid at a concentration range of about 400-600 μg/mland basic fibroblast growth factor (bFGF) at a concentration range ofabout 50-200 ng/ml.

According to some embodiments of the invention the culture medium theculture medium which comprises ascorbic acid at a concentration range ofabout 400-600 μg/ml and basic fibroblast growth factor (bFGF) at aconcentration range of about 50-200 ng/ml is capable of maintainingpluripotent stem cells in an undifferentiated state in the absence offeeder cell support.

According to some embodiments of the invention, the concentration ofascorbic acid in the culture medium is between about 410 μg/ml to about590 μg/ml, between about 420 μg/ml to about 580 μg/ml, between about 450μg/ml to about 550 μg/ml, between about 460 μg/ml to about 540 μg/ml,between about 470 μg/ml to about 530 μg/ml, between about 490 μg/ml toabout 520 μg/ml, e.g., between about 490 μg/ml to about 510 μg/ml. e.g.,about 500 μg/ml.

According to some embodiments of the invention, the concentration ofbFGF in the culture medium is between about 50 ng/ml to about 200 ng/ml,between about 60 ng/ml to about 190 ng/ml, between about 70 ng/ml toabout 180 ng/ml, between about 80 ng/ml to about 170 ng/ml, betweenabout 90 ng/ml to about 160 ng/ml, between about 90 ng/ml to about 150ng/ml, between about 90 ng/ml to about 130 ng/ml, between about 90 ng/mlto about 120 ng/ml. e.g., about 100 ng/ml.

According to some embodiments of the invention, the concentration ofbFGF in the culture medium is about 50, about 55, about 60, about 65,about 70, about 80, about 85, about 90, about 95, about 100, about 105,about 110, about 115, about 120, about 125, about 130, about 135, about140, about 145, about 150, about 160, about 165, about 170, about 175,about 180, about 185, about 190, about 195, about 200 ng/ml.

According some embodiments of the invention the culture medium whichcomprises ascorbic acid at a concentration range of about 400-600 μg/mland basic fibroblast growth factor (bFGF) at a concentration range ofabout 50-200 ng/ml, further comprises xeno-free serum replacement.

According to some embodiments of the invention, the culture medium whichcomprises ascorbic acid at a concentration range of about 400-600 μg/mland basic fibroblast growth factor (bFGF) at a concentration range ofabout 50-2000 ng/ml, further comprises a lipid mixture.

According to some embodiments of the invention, the culture mediumcomprises bFGF at a concentration of about 50-200 ng/ml and ascorbicacid at a concentration of about 400-600 μg/ml is devoid ofsodium-bicarbonate.

According to some embodiments of the invention, the culture mediumcomprises bFGF at a concentration of about 50-200 ng/ml and ascorbicacid at a concentration of about 400-600 μg/ml, xeno-free serumreplacement at a concentration of about 1% and lipid mixture at aconcentration of about 1%.

A non-limiting example of a xeno-free, serum-free, and TGFβ isoform-freeculture medium which comprises ascorbic acid at a concentration range ofabout 400-600 μg/ml, bFGF at a concentration range of about 50-200 ng/m,xeno-free serum replacement and a lipid mixture and which is capable ofmaintaining pluripotent stem cells such as hESCs and human iPS cells ina proliferative and undifferentiated state for at least 21 passages inthe absence of feeder cell support is the HA77 culture medium (Example 1of the Examples section which follows) or a culture medium similar tothe HA77 medium but which is devoid of sodium bi-carbonate such as aculture medium which consists of DMEM/F12 (94%) (Biological Industries,Israel, Sigma Israel), L-glutamine 2 mM (Invitrogen corporation. Sigma,Israel), ascorbic acid 500 μg/ml (Sigma, Israel), bFGF—100 ng(Invitrogen corporation), SR3—1% (Sigma, Israel), and defined lipidmixture 1% (Invitrogen corporation, Sigma, Israel). The presentinventors have uncovered novel serum-free and highly defined culturemedia, which can maintain pluripotent stem cells in a proliferative,pluripotent and undifferentiated state in two-dimensional andthree-dimensional (i.e., a suspension culture) systems in the absence offeeder cell support.

As used herein the phrase “suspension culture” refers to a culture inwhich the pluripotent stem cells are suspended in a medium rather thanadhering to a surface.

According to some embodiments of the invention the serum-free culturemedium which can maintain pluripotent stem cells in a proliferative,pluripotent and undifferentiated state in two-dimensional andthree-dimensional culture systems in the absence of feeder cell supportcomprises basic fibroblast growth factor (bFGF) at a concentration rangeof about 50-200 ng/ml.

According to some embodiments of the invention the culture mediumcomprises between about 55-190 ng/ml. e.g., between about 60-190 ng/ml,e.g., between about 70-180 ng/ml. e.g., between about 80-160 ng/ml.e.g., between about 90-150 ng/ml, e.g., between about 90-140 ng/ml,e.g., between about 90-130 ng/ml. e.g., between about 90-120 ng/ml,e.g., between about 90-110 ng/ml, e.g., between about 95-105 ng/ml,e.g., about 100 ng/ml.

According to some embodiments of the invention the culture medium whichcomprises bFGF between about 50-200 ng/ml further comprises serumreplacement.

A non-limiting example of a culture medium which comprises bFGF at aconcentration between about 50-200 ng/ml is the YF100 medium whichcomprises a basic medium (e.g., DMEM/F12, 85%), serum replacement (15%),bFGF (100 ng/ml). L-glutamine (2 mM), β-mercaptoethanol (0.1 mM) andnon-essential amino acid stock (1%).

According to some embodiments of the invention the serum-free culturemedium which can maintain pluripotent stem cells in a proliferative,pluripotent and undifferentiated state in two-dimensional andthree-dimensional culture systems in the absence of feeder cell supportconsists of a basic medium, ascorbic acid at a concentration range ofabout 50 μg/ml to about 500 μg/ml, bFGF at a concentration range betweenabout 2 ng/ml to about 20 ng/ml. L-glutamine, and serum replacement.

According to some embodiments of the invention the serum-free culturemedium which can maintain pluripotent stem cells in a proliferative,pluripotent and undifferentiated state in two-dimensional andthree-dimensional culture systems in the absence of feeder cell supportconsists of a basic medium, ascorbic acid at a concentration range ofabout 50 μg/ml to about 500 μg/ml, bFGF at a concentration range betweenabout 2 ng/ml to about 20 ng/ml, L-glutamine, serum replacement and alipid mixture.

According to some embodiments of the invention the concentration ofascorbic acid is about 50 μg/ml.

According to some embodiments of the invention the concentration ofascorbic acid is about 500 μg/ml.

According to some embodiments of the invention the concentration of bFGFis about 4 ng/ml.

The basic medium can be any known tissue culture medium such as DMEM/F12(Biological Industries, Israel, or Sigma Israel), Ko-DMEM (Invitrogen).

The concentration of the basic medium depends on the concentration ofthe other medium ingredients such as the serum replacement.

The serum replacement can be any xeno-free serum replacement (devoid ofanimal contaminants) at a concentration range from 1-20% depending onthe serum replacement used. For example, if the SR3 serum replacement isused then it concentration in the medium is about 1%.

According to some embodiments of the invention the concentration ofL-glutamine is about 2 mM.

According to some embodiments of the invention the concentration of thelipid mixture (Sigma, Israel; or Invitrogen, Israel) is about 1%.

Non-limiting examples of such a culture medium include the modifiedHA13(a) medium [DMEM/F12 (95%). L-glutamine 2 mM, ascorbic acid 500μg/ml, bFGF—4 ng, and SR3—1%]; the modified HA13(b) medium [DMEM/F12(95%), L-glutamine 2 mM, ascorbic acid 500 μg/ml, bFGF—4 ng, SR3—1% anda lipid mixture (1%)]; the modified HA13(c) medium [DMEM/F12 (95%).L-glutamine 2 mM, ascorbic acid 50 μg/ml, bFGF—4 ng, and SR3—1% J; andthe modified HA13(d) medium [DMEM/F12 (95%), L-glutamine 2 mM, ascorbicacid 50 μg/ml, bFGF—4 ng, SR3—1% and a lipid mixture (1%)]. Theseculture media were capable of maintaining pluripotent stem cells (e.g.,hESCs and hips cells) in a proliferative, pluripotent andundifferentiated state for at least 20 passages when cultured in atwo-dimensional (e.g., on a feeder-layer free culture system; data notshown) and for at least 20 passages when cultured on a three-dimensionalculture system (e.g., suspension culture without adherence to anexternal substrate, cell encapsulation or to protein carrier; data notshown).

According to some embodiments of the invention the serum-free culturemedium which can maintain pluripotent stem cells in a proliferative,pluripotent and undifferentiated state in two-dimensional andthree-dimensional culture systems in the absence of feeder cell supportcomprises an IL6RIL6 chimera at a concentration range of about 50-200picogram per milliliter (pg/ml).

As used herein the phrase “IL6RIL6 chimera” refers to a chimericpolypeptide which comprises the soluble portion of interleukin-6receptor [IL-6-R, e.g., the human IL-6-R as set forth by GenBankAccession No. AAH89410; SEQ ID NO:33; e.g., a portion of the soluble IL6receptors as set forth by amino acids 112-355 (SEQ ID NO:34) of GenBankAccession No. AAH89410] and the interleukin-6 (IL6; e.g., human IL-6 asset forth by GenBank Accession No. CAG29292; SEQ ID NO:35) or abiologically active fraction thereof (e.g., a receptor binding domain).

It should be noted that when constructing the IL6RIL6 chimera the twofunctional portions (i.e., the IL6 and its receptor) can be directlyfused (e.g., attached or translationally fused, i.e., encoded by asingle open reading frame) to each other or conjugated (attached ortranslationally fused) via a suitable linker (e.g., a polypeptidelinker). According to some embodiments of the invention, the IL6RIL6chimeric polypeptide exhibits a similar amount and pattern ofglycosylation as the naturally occurring IL6 and IL6 receptor. Forexample, a suitable IL6RIL6 chimera is as set forth in SEQ ID NO:36 andin FIG. 11 of WO 99/02552 to Revel M., et al., which is fullyincorporated herein by reference.

It will be appreciated that any of the proteinaceous factors used in theculture medium of the present invention (e.g., the IL6RIL6 chimera,bFGF, TGFβ₃) can be recombinantly expressed or biochemicallysynthesized. In addition, naturally occurring proteinaceous factors suchas bFGF and TGFβ can be purified from biological samples (e.g., fromhuman serum, cell cultures) using methods well known in the art.

Biochemical synthesis of the proteinaceous factors of the presentinvention (e.g., the IL6RIL6 chimera) can be performed using standardsolid phase techniques. These methods include exclusive solid phasesynthesis, partial solid phase synthesis methods, fragment condensationand classical solution synthesis.

Recombinant expression of the proteinaceous factors of the presentinvention (e.g., the IL6RIL6 chimera) can be generated using recombinanttechniques such as described by Bitter et al., (1987) Methods inEnzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol.185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al.(1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680,Brogli et al., (1984) Science 224:838-843, Gurley et al. (1986) Mol.Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988. Methods for PlantMolecular Biology, Academic Press, NY, Section VIII, pp 421-463.Specifically, the IL6RIL6 chimera can be generated as described in PCTpublication WO 99/02552 to Revel M., et al, and Chebath J., et al.,1997, which are fully incorporated herein by reference.

According to some embodiments of the invention, the concentration of theIL6RIL6 chimera in the culture medium is in the range from about 55μg/ml to about 195 μg/ml, e.g., from about 60 μg/ml to about 190 μg/mle.g., from about 65 μg/ml to about 185 μg/ml, e.g., from about 70 μg/mlto about 180 μg/ml, e.g., from about 75 μg/ml to about 175 μg/ml, e.g.,from about 80 μg/ml to about 170 μg/ml. e.g., from about 85 μg/ml toabout 165 μg/ml, e.g., from about 90 μg/ml to about 150 μg/ml. e.g.,from about 90 μg/ml to about 140 μg/ml, e.g., from about 90 μg/ml toabout 130 μg/ml, e.g., from about 90 μg/ml to about 120 μg/ml, e.g.,from about 90 μg/ml to about 110 μg/ml, e.g., from about 95 μg/ml toabout 105 μg/ml. e.g., from about 98 μg/ml to about 102 μg/ml, e.g.,about 100 μg/ml.

According to some embodiments of the invention, the IL6RIL6chimera-containing culture medium further comprises bFGF.

According to some embodiments of the invention, concentration of bFGF inthe IL6RIL6 chimera-containing culture medium is in the range of fromabout 1 ng/ml to about 10 μg/ml. e.g., from about 2 ng/ml to about 1μg/ml. e.g., from about 2 ng/ml to about 500 ng/ml, e.g., from about 5ng/ml to about 150 ng/ml, e.g., from about 5 ng/ml to about 100 ng/ml,e.g., from about 5 ng/ml to about 80 ng/ml, e.g., from about 5 ng/ml toabout 50 ng/ml, e.g., from about 5 ng/ml to about 30 ng/ml. e.g., about5 ng/ml. e.g., about 10 ng/ml, e.g., about 15 ng/ml, e.g., about 20ng/ml.

According to some embodiments of the invention, the IL6RIL6chimera-containing culture medium further comprises serum replacement.

According to some embodiments of the invention, the concentration ofKNOCKOUT™ Serum Replacement in the IL6RIL6 chimera-containing culturemedium is in the range from about 1% (v/v) to about 50% (v/v). e.g.,from about 5% (v/v) to about 40% (v/v), e.g., from about 5% (v/v) toabout 30% (v/v). e.g., from about 10% (v/v) to about 30% (v/v), e.g.,from about 10% (v/v) to about 25% (v/v), e.g., from about 10% (v/v) toabout 20% (v/v), e.g., about 15% (v/v).

According to some embodiments of the invention, the culture mediumcomprises IL6RIL6 chimera at a concentration range of about 50-200μg/ml, bFGF at a concentration range of about 5-50 ng/ml and serumreplacement at a concentration of about 5-40%.

For example, as is shown in Example 4 of the Examples section whichfollows, the CM100Fp culture medium was shown capable of maintainingpluripotent stem cells such as hESCs and human iPS cells in aproliferative, pluripotent and undifferentiated state for at least 50passages in a suspension culture devoid of substrate adherence.

According to some embodiments of the invention, the serum-free culturemedium which can maintain pluripotent stem cells in a proliferative,pluripotent and undifferentiated state in two-dimensional andthree-dimensional culture systems in the absence of feeder-cells supportcomprises LIF at a concentration of at least 2000 units/ml.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine which isinvolved in the induction of hematopoietic differentiation, induction ofneuronal cell differentiation, regulator of mesenchymal to epithelialconversion during kidney development, and may also have a role in immunetolerance at the maternal-fetal interface. The LIF used in the culturemedium of some embodiments of the invention can be a purified, syntheticor recombinantly expressed LIF protein [e.g., human LIF polypeptideGenBank Accession No. NP_002300.1 (SEQ ID NO:37); human LIFpolynucleotide GenBank Accession No. NM_002309.3 (SEQ ID NO:38). Itshould be noted that for the preparation of a xeno-free culture mediumLIF is preferably purified from a human source or is recombinantlyexpressed. Recombinant human LIF can be obtained from various sourcessuch as Chemicon, USA (Catalogue No. LIF10100) and AbD Serotec(MorphoSys US Inc, Raleigh, N.C. 27604, USA). Murine LIF ESGRO® (LIF)can be obtained from Millipore, USA (Catalogue No. ESG1107).

According to some embodiments of the invention, the concentration of LIFin the culture medium is from about 2000 units/ml to about 10.000units/ml. e.g., from about 2000 units/ml to about 8,000 units/ml, e.g.,from about 2000 units/ml to about 6,000 units/ml, e.g., from about 2000units/ml to about 5,000 units/ml. e.g., from about 2000 units/ml toabout 4,000 units/ml.

According to some embodiments of the invention, the concentration of LIFin the culture medium is at least about 2000 units/ml, e.g., at leastabout 2100 units/ml, e.g., at least about 2200 units/ml. e.g., at leastabout 230 units/ml, e.g., at least about 2400 units/mli e.g., at leastabout 2500 units/ml, e.g., at least about 2600 units/ml, e.g., at leastabout 2700 units/ml, e.g., at least about 2800 units/ml, e.g., at leastabout 2900 units/ml, e.g., at least about 2950 units/ml, e.g., about3000 units/ml.

According to some embodiments of the invention, the LIF-containingculture medium further comprises bFGF.

The concentration of bFGF in the LIF-containing culture medium is in therange of about 0.1 ng/ml to about 10 μg/ml. e.g., from about 2 ng/ml toabout 1 μg/ml, e.g., from about 2 ng/ml to about 500 ng/ml. e.g., fromabout 5 ng/ml to about 150 ng/ml. e.g., from about 5 ng/ml to about 100ng/ml, e.g., from about 5 ng/ml to about 80 ng/ml, e.g., from about 5ng/ml to about 50 ng/ml. e.g., from about 5 ng/ml to about 30 ng/ml,e.g., about 5 ng/ml, e.g., about 10 ng/ml, e.g., about 15 ng/ml, e.g.,about 20 ng/ml.

According to some embodiments of the invention, the LIF-containingculture medium further comprises serum replacement.

According to some embodiments of the invention, the culture mediumcomprises LIF at a concentration of about 2000-10.000 units/ml, bFGF ata concentration range from about 0.1 ng/ml to about 10 μg/ml andKNOCKOUT™ Serum Replacement at a concentration range from about 1% (v/v)to about 50% (v/v).

According to some embodiments of the invention, the culture mediumcomprises LIF at a concentration of about 2000-5.000 units/ml, bFGF at aconcentration of about 5-50 ng/ml and serum replacement at aconcentration of about 5-30%.

For example, as shown in Example 4 of the Examples section whichfollows, the yFL3 culture medium was shown capable of maintainingpluripotent stem cells such as human ESCs and human iPS cells in aproliferative, pluripotent and undifferentiated state for at least 10passages when cultured in a suspension culture.

According to some embodiments of the invention, the ingredients includedin the culture medium of some embodiments of the invention aresubstantially pure, with a tissue culture and/or a clinical grade.

According to an aspect of some embodiments of the invention there isprovided a cell culture which comprises the pluripotent stem cell ofsome embodiments of the invention and the culture medium of someembodiments of the invention.

According to an aspect of some embodiments of the invention cell cultureis feeder cells free (e.g., being devoid of feeder cells or feeder cellconditioned medium).

According to some embodiments of the invention the pluripotent stemcells which are included in the cell culture of some embodiments of theinvention exhibit a stable karyotype (chromosomal stability) during theculturing period, e.g., for at least 2 passages, e.g., at least 4passages, e.g., at least 8 passages, e.g., at least 15 passages, e.g.,at least 20 passages, e.g., at least 25 passages, e.g., at least 30passages. e.g., at least 35 passages, e.g., at least 40 passages, e.g.,at least 45 passages, e.g., at least 50 passages.

According to some embodiments of the invention, the cell culture of theinvention exhibit a doubling time of at least 20 hours, e.g., a doublingtime which is between 20 to 40 hours (e.g., about 36 hours), thusrepresenting a non-tumorigenic, genetically stable pluripotent stemcells (e.g., hESCs and iPS cells).

According to some embodiments of the invention, the cell culture of theinvention is characterized by at least 40%, at least 50%, at least 60%,e.g., at least 70%, e.g., at least 80%, e.g., at least 85%, e.g., atleast 90%. e.g., at least 95% of undifferentiated pluripotent stemcells.

According to an aspect of some embodiments of the invention, there isprovided a method of expanding and maintaining pluripotent stem cells ina pluripotent and undifferentiated state.

According to some embodiments of the invention, the method of expandingand maintaining pluripotent stem cells in an undifferentiated state iseffected by culturing the pluripotent stem cells in any of the novelculture media of the invention (described herein).

According to some embodiments of the invention, the method of expandingand maintaining pluripotent stem cells in an undifferentiated state iseffected by culturing the pluripotent stem cells in a culture mediumbeing serum-free, feeder-free, matrix-free and protein carrier-free andcomprising basic fibroblast growth factor (bFGF) at a concentrationrange of about 50-200 ng/ml.

According to some embodiments of the invention culturing is effected ona two-dimensional culture system such as a matrix or a feeder celllayer.

For example, culturing on a two-dimensional culture system can beperformed by plating the pluripotent stem cells onto a matrix or afeeder cell layer in a cell density which promotes cell survival andproliferation but limits differentiation. Typically, a plating densityof between about 15,000 cells/cm² and about 3,00000 cells/cm² is used.

It will be appreciated that although single-cell suspensions ofpluripotent stem cells are usually seeded, small clusters may also beused. To this end, enzymatic digestion (such as with type IVcollagenase) utilized for cluster disruption (see “General Materials andExperimental Methods” in the Examples section which follows) isterminated before stem cells become completely dispersed and the cellsare triturated with a pipette such that clumps (i.e., 10-200 cells) areformed. However, measures are taken to avoid large clusters which maycause cell differentiation.

As used herein, the term “matrix” refers to any substance to which thepluripotent stem cells can adhere and which therefore can substitute thecell attachment function of feeder cells. Such a matrix typicallycontains extracellular components to which the pluripotent stem cellscan attach and thus it provides a suitable culture substrate.

According to some embodiments of the invention the matrix comprises anextracellular matrix.

The extracellular matrix can be composed of components derived frombasement membrane or extracellular matrix components that form part ofadhesion molecule receptor-ligand couplings. MATRIGEL® (BectonDickinson. USA) is one example of a commercially available matrix whichis suitable for use with the present invention. MATRIGEL® is a solublepreparation from Engelbreth-Holm-Swarm tumor cells that gels at roomtemperature to form a reconstituted basement membrane; MATRIGEL® is alsoavailable as a growth factor reduced preparation. Other extracellularmatrix components and component mixtures which are suitable for use withthe present invention include foreskin matrix, laminin matrix,fibronectin matrix, proteoglycan matrix, entactin matrix, heparansulfate matrix, collagen matrix and the like, alone or in variouscombinations thereof.

According to some embodiments of the invention the matrix is xeno-free.

In cases where complete animal-free culturing conditions are desired,the matrix is preferably derived from a human source or synthesizedusing recombinant techniques such as described hereinabove. Suchmatrices include, for example, human-derived fibronectin, recombinantfibronectin, human-derived laminin, foreskin fibroblast matrix or asynthetic fibronectin matrix. Human derived fibronectin can be fromplasma fibronectin or cellular fibronectin, both of which can beobtained from Sigma. St. Louis, Mo., USA. Human derived laminin andforeskin fibroblast matrix can be obtained from Sigma. St. Louis, Mo.USA. A synthetic fibronectin matrix can be obtained from Sigma. St.Louis, Mo. USA.

According to some embodiments of the invention, culturing is effected ona feeder cell layer.

According to some embodiments of the invention, the method of expandingand maintaining pluripotent stem cells in an undifferentiated state iseffected by culturing the pluripotent stem cells on a feeder cell layerin a serum-free and xeno-free culture medium which comprises basicfibroblast growth factor (bFGF), transforming growth factor beta-3(TGFβ3) and ascorbic acid, wherein a concentration of the ascorbic acidin the culture medium is at least 50 g/ml.

According to some embodiments of the invention, the method of expandingand maintaining pluripotent stem cells in an undifferentiated state iseffected by culturing the pluripotent stem cells on a feeder cell layerin a serum-free and xeno-free culture medium which comprises ascorbicacid at a concentration range of about 400-600 μg/ml, basic fibroblastgrowth factor (bFGF) at a concentration range of about 50-200 ng/ml,xeno-free serum replacement and a lipid mixture.

According to some embodiments of the invention, the feeder cell layer isxeno-free.

According to some embodiments of the invention, the feeder cell layer isa foreskin fibroblasts feeder cell layer.

According to some embodiments of the invention, culturing according tosome embodiments of the invention is effected in a suspension culture.

According to some embodiments of the invention, the suspension cultureis devoid of substrate adherence, e.g., without adherence to an externalsubstrate such as components of extracellular matrix, a glassmicrocarrier or beads.

According to some embodiments of the invention, culturing of thepluripotent stem cells in a suspension culture is effected in a proteincarrier-free culture medium.

As used herein the phrase “protein carrier” refers to a protein whichacts in the transfer of proteins or nutrients (e.g., minerals such aszinc) to the cells in the culture.

Such protein carriers can be, for example, albumin (e.g., bovine serumalbumin), Albumax (lipid enriched albumin) or plasmanate (human plasmaisolated proteins). Since these carriers are derived from either humanor animal sources their use in hESCs of human iPS cell cultures islimited by batch-specific variations and/or exposure to pathogens. Thus,a culture medium which is devoid of a protein carrier (e.g., albumin) ishighly advantageous since it enables a truly defined medium that can bemanufacture from recombinant or synthetic materials.

According to some embodiments of the invention, culturing of thepluripotent stem cells in a suspension culture is effected in aserum-free and feeder cell-free culture medium.

It should be noted that some protocols of culturing pluripotent stemcells such as hESCs and iPS cells include microencapsulation of thecells inside a semipermeable hydrogel membrane, which allows theexchange of nutrients, gases, and metabolic products with the bulkmedium surrounding the capsule (for details see e.g., U.S. PatentApplication No. 20090029462 to Beardsley et al.).

According to some embodiments of the invention, the pluripotent stemcells cultured in the suspension culture are devoid of cellencapsulation.

According to an aspect of some embodiments of the invention, there isprovided a method of expanding induced pluripotent stem (iPS) cells andmaintaining the iPS cells in an undifferentiated state. The method iseffected by culturing the iPS cells in a suspension culture underculturing conditions devoid of substrate adherence and devoid of cellencapsulation and which allow expansion of the iPS cells in theundifferentiated state.

According to some embodiments of the invention, culturing of thepluripotent stem cells in a suspension culture is effected in thepresence of the IL6RIL6 chimera-containing culture medium in which theconcentration of the IL6RIL6 chimera is in the range of about 50-200picograms per milliliter (μg/ml).

According to some embodiments of the invention, culturing of thepluripotent stem cells in a suspension culture is effected in thepresence of the leukemia inhibitory factor (LIF)-containing culturemedium in which the concentration of LIF is at least about 2000units/ml.

According to some embodiments of the invention, culturing of thepluripotent stem cells in a suspension culture is effected in thepresence of a medium which comprises basic fibroblast growth factor(bFGF) at a concentration range of about 50 ng/ml to about 200 ng/ml,e.g., between about 60 ng/ml to about 190 ng/ml, e.g., between about 70ng/ml to about 180 ng/ml, e.g., between about 80 ng/ml to about 170ng/ml, e.g., between about 90 ng/ml to about 160 ng/ml, e.g., betweenabout 90 ng/ml to about 150 ng/ml, e.g., between about 90 ng/ml to about130 ng/ml, e.g., between about 90 ng/ml to about 120 ng/ml. e.g., about100 ng/ml.

For example, a non-limiting example of a medium which was found suitablefor culturing hESCs and human iPS cells in a suspension culture devoidof substrate adherence and cell encapsulation is the yF100 medium whichcomprises serum replacement and 100 ng/ml bFGF.

According to some embodiments of the invention, culturing of thepluripotent stem cells in a suspension culture is effected in thepresence of a medium which comprises the IL6RIL6 chimera at aconcentration range of about 50-200 nanogram per milliliter (ng/ml) andbFGF at a concentration in the range of 1-50 ng/ml.

For example, a non-limiting example of a medium which was found suitablefor culturing hESCs and human iPS cells in a suspension culture devoidof substrate adherence and cell encapsulation is the CM100F medium whichcomprises serum replacement, the IL6RIL6 chimera at a concentration of100 ng/ml and bFGF at a concentration of 10 ng/ml.

For example, using the CM100Fp, CM100F, yF100 or yFL3 culture media thepresent inventors expanded pluripotent stem cells in a suspensionculture in a proliferative, pluripotent and undifferentiated state forat least 50 passages (see e.g., FIGS. 3A-C, 4, 5A-C and 6A-D and isdescribed in Examples 4 and 5 of the Examples section which follows).

Culturing in a suspension culture according to the method of someembodiments of the invention is effected by plating the pluripotent stemcells in a culture vessel at a cell density which promotes cell survivaland proliferation but limits differentiation. Typically, a platingdensity of between about 5×10⁴-2×10⁶ cells per ml is used. It will beappreciated that although single-cell suspensions of stem cells areusually seeded, small clusters such as 10-200 cells may also be used.

In order to provide the pluripotent stem cells with sufficient andconstant supply of nutrients and growth factors while in the suspensionculture, the culture medium can be replaced on a daily basis, or, at apre-determined schedule such as every 2-3 days. For example, replacementof the culture medium can be performed by subjecting the pluripotentstem cells suspension culture to centrifugation for about 3 minutes at80 g. and resuspension of the formed pluripotent stem cells pellet in afresh medium. Additionally or alternatively, a culture system in whichthe culture medium is subject to constant filtration or dialysis so asto provide a constant supply of nutrients or growth factors to thepluripotent stem cells may be employed.

Since large clusters of pluripotent stem cells may cause celldifferentiation, measures are taken to avoid large pluripotent stemcells aggregates. According to some embodiments of the invention, theformed pluripotent stem cells clumps are dissociated every 5-7 days andthe single cells or small clumps of cells are either split intoadditional culture vessels (i.e., passaged) or remained in the sameculture vessel yet with additional culture medium. For dissociation oflarge pluripotent stem cells clumps, a pellet of pluripotent stem cells(which may be achieved by centrifugation as described hereinabove) or anisolated pluripotent stem cells clump can be subject to enzymaticdigestion and/or mechanical dissociation.

Enzymatic digestion of pluripotent stem cells clump(s) can be performedby subjecting the clump(s) to an enzyme such as type IV Collagenase(Worthington biochemical corporation. Lakewood, N.J., USA) and/orDispase (Invitrogen Corporation products. Grand Island N.Y., USA). Thetime of incubation with the enzyme depends on the size of cell clumpspresent in the suspension culture. Typically, when pluripotent stemcells cell clumps are dissociated every 5-7 days while in the suspensionculture, incubation of 20-60 minutes with 1.5 mg/ml type IV Collagenaseresults in small cell clumps which can be further cultured in theundifferentiated state. Alternatively, pluripotent stem cells clumps canbe subjected to incubation of about 25 minutes with 1.5 mg/ml type IVCollagenase followed by five minutes incubation with 1 mg/ml Dispase. Itshould be noted that passaging of human ESCs with trypsin may result inchromosomal instability and abnormalities (see for example, Mitalipova MM., et al., Nature Biotechnology, 23: 19-20, 2005 and Cowan C A et al.,N. Engl. J. of Med. 350: 1353-1356, 2004). According to some embodimentsof the invention, passaging hESC or iPS cell with trypsin should beavoided.

Mechanical dissociation of large pluripotent stem cells clumps can beperformed using a device designed to break the clumps to a predeterminedsize. Such a device can be obtained from CellArtis Goteborg, Sweden.Additionally or alternatively, mechanical dissociation can be manuallyperformed using a needle such as a 27 g needle (BD Microlance, Drogheda,Ireland) while viewing the clumps under an inverted microscope.

According to some embodiments of the invention, following enzymatic ormechanical dissociation of the large cell clumps, the dissociatedpluripotent stem cells clumps are further broken to small clumps using200 μl Gilson pipette tips (e.g., by pipetting up and down the cells).

The culture vessel used for culturing the pluripotent stem cells insuspension according to the method of some embodiments of the inventioncan be any tissue culture vessel (e.g., with a purity grade suitable forculturing pluripotent stem cells) having an internal surface designedsuch that pluripotent stem cells cultured therein are unable to adhereor attach to such a surface (e.g., non-tissue culture treated cells, toprevent attachment or adherence to the surface). Preferably, in order toobtain a scalable culture, culturing according to some embodiments ofthe invention is effected using a controlled culturing system(preferably a computer-controlled culturing system) in which cultureparameters such as temperature, agitation, pH, and pO₂ is automaticallyperformed using a suitable device. Once the culture parameters arerecorded, the system is set for automatic adjustment of cultureparameters as needed for pluripotent stem cells expansion.

As described in the Examples section which follows, the pluripotent stemcells were cultured under dynamic conditions (i.e., under conditions inwhich the pluripotent stem cells are subject to constant movement whilein the suspension culture; see e.g., FIGS. 6A-D; Example 5) or undernon-dynamic conditions (i.e., a static culture; see e.g., FIGS. 3A-C, 4and 5A-C; Example 4) while preserving their, proliferative, pluripotentcapacity and karyotype stability for at least 30 passages.

For non-dynamic culturing of pluripotent stem cells, the pluripotentstem cells can be cultured in uncoated 58 mm Petri dishes (Greiner,Frickenhausn, Germany).

For dynamic culturing of pluripotent stem cells, the pluripotent stemcells can be cultured in spinner flasks [e.g., of 200 ml to 1000 ml, forexample 250 ml which can be obtained from CellSpin of IntegraBiosciences. Fernwald, Germany; of 100 ml which can be obtained fromBellco, Vineland, N.J.; or in 125 ml Erlenmeyer (Corning Incorporated,Corning N.Y., USA)] which can be connected to a control unit and thuspresent a controlled culturing system. The culture vessel (e.g., aspinner flask, an Erlenmeyer) is shaken continuously. According to someembodiments of the invention the culture vessels are shaken at 90 roundsper minute (rpm) using a shaker (S3.02.10L, ELMI ltd, Riga. Latvia).According to some embodiments of the invention the culture medium ischanged daily.

According to some embodiments of the invention, when cultured accordingto the teachings of the present invention, the growth of the pluripotentstem cells is monitored to determine their differentiation state. Thedifferentiation state can be determined using various approachesincluding, for example, morphological evaluation (e.g., as shown inFIGS. 1A-C, 3A-C) and/or detection of the expression pattern of typicalmarkers of the undifferentiated state using immunological techniquessuch as flow cytometry for membrane-bound markers, immunohistochemistryor immunofluorescence for extracellular and intracellular markers andenzymatic immunoassay, for secreted molecular markers. For example,immunofluorescence employed on hESCs or human iPS cells culturedaccording to the method of some embodiments of the invention revealedthe expression of Oct4, stage-specific embryonic antigen (SSEA) 4, thetumor-rejecting antigen (TRA)-1-60 and TRA-1-81 (FIGS. 2A-C, 5A-C and6A-D). Additionally, the level of transcripts of specificundifferentiation markers (e.g., Oct 4, Nanog, Sox2, Rex1, Cx43, FGF4)or differentiation markers (e.g., albumin, glucagons, α-cardiac actin,β-globulin, Flk1, AC133 and neurofilament) can be detected usingRNA-based techniques such as RT-PCR analysis and/or cDNA microarrayanalysis.

Determination of ES cell differentiation can also be effected viameasurements of alkaline phosphatase activity. Undifferentiated human EScells have alkaline phosphatase activity which can be detected by fixingthe cells with 4% paraformaldehyde and developing with the Vector Redsubstrate kit according to manufacturer's instructions (VectorLaboratories, Burlingame, Calif., USA).

The present inventors have uncovered that the novel xeno-free and serumfree culture media of the invention can be used to derive newpluripotent stem cell lines.

Thus, as is further shown in the Examples section which follows, usingthe HA40/4 medium culture medium the present inventors were capable ofderiving a new hESC line referred to as “WC1” from whole blastocystscultured on human foreskin fibroblasts feeder layer (Example 3 of theExamples section which follows).

The term “deriving” as used herein refers to generating an embryonicstem cell line or an induced pluripotent stem cell line from at leastone embryonic stem or induced pluripotent cell.

According to some embodiments of the invention, the pluripotent stemcell line is an embryonic stem cell line, and the method of deriving theembryonic stem cell line is effected by: (a) obtaining an embryonic stemcell from a pre-implantation stage blastocyst, post-implantation stageblastocyst and/or a genital tissue of a fetus; and (b) culturing theembryonic stem cell in the culture medium of some embodiments of theinvention, thereby deriving the embryonic stem cell line.

As used herein the phrase “embryonic stem cell line” refers to embryonicstem cells which are derived from a single or a group of embryonic stemcells of a single organism (e.g., a single human blastocyst), and whichare characterized by the ability to proliferate in culture whilemaintaining the undifferentiated state and the pluripotent capacity.

Obtaining an embryonic stem cell from a pre-implantation stageblastocyst, post-implantation stage blastocyst and/or a genital tissueof a fetus can be performed using methods known in the art, as describedhereinabove and in Example 3 of the Examples section which follows.Briefly, the zona pellucida is removed from a 5-7 day-old blastocystusing Tyrode's acidic solution (Sigma, St Louis Mo., USA), thetrophoblast layer is specifically removed either by immunosurgery ormechanically using 27 g needles and the exposed ICM is either directlycultured in a suitable culture system (e.g., feeder layers, feeder-freematrix or a suspension culture) in the presence of any of the culturemedia described hereinabove for 4-10 days (in case a preimplantationblastocyst is used) or subject to in vitro implantation by culturing theICM for 6-8 days (to obtain cells of a 13 day-old blastocyst in case apost-implantation/pre-gastrulation blastocyst is used) on feeder layersor a feeder-free culturing system which allow implantation of theblastocyst to the surface, following which the implanted cells areisolated and can be further cultured on feeder layers, feeder-freematrix or a suspension culture in the presence of any of the culturemedia described hereinabove as described hereinunder. When using thegenital tissue of a fetus, the genital ridges are dissociated and cutinto small chunks which are thereafter disaggregated into cells bymechanical dissociation. The single cell EG cells are then cultured inany of the culture media described hereinabove for 4-10 days.

According to some embodiments of the invention, the pluripotent stemcell line is an induced pluripotent stem cell (iPS cell) line, and themethod of deriving the iPS cell line is effected by: (a) inducing asomatic cell to a pluripotent stem cell; and (b) culturing thepluripotent stem cell in the culture medium of some embodiments of theinvention, thereby deriving the induced pluripotent stem cell line.

As used herein the phrase “induced pluripotent stem cell line” refers topluripotent stem cells derived from a single induced pluripotent stemcell, which are characterized by the ability to proliferate in culturewhile maintaining the undifferentiated state and the pluripotentcapacity.

Methods of inducing pluripotent stem cells are well known in the art andexamples are given in Takahashi and Yamanaka, 2006; Takahashi et al,2007; Meissner et al, 2007; Okita et al, 2007, Yu et al. 2007; Nakagawaet al, 2008, Yu J. et al., Science. 2009, 324: 797-801; Park et al,2008; Hanna et al, 2007; Lowry et al, 2008; Aoi et al. 2008; all ofwhich are fully incorporated by reference herein.

Once obtained the ESCs of iPS cells are further cultured in any of theculture media described hereinabove which allow expansion of thepluripotent stem cells in the undifferentiated state, essentially asdescribed hereinabove.

It will be appreciated that an established pluripotent stem cell line(e.g., embryonic stem cell line or induced pluripotent stem cell line)can be subject to freeze/thaw cycles without hampering the proliferativecapacity of the cells in the undifferentiated state while preservingtheir pluripotent capacity. For example, as is shown in the Examplessection which follows, using 15% serum replacement and 10% DMSO, hESCsor human iPS cells were successfully frozen and thawed.

As described in Examples 1, 2, 4 and 5 of the Examples section whichfollows, hESCs and human iPS cells which were expanded and maintained inany of the culture media described hereinabove are pluripotent (i.e.,capable of differentiating into all cell types of the three embryonicgerm layers, the ectoderm, the endoderm and the mesoderm) as evidencedin vitro (by the formation of EBs) and in vivo (by the formation ofteratomas) after a prolonged culture period (e.g., of at least 20 or 30passages) in the two-dimensional (e.g., feeder-free matrices or foreskinfeeders) or three-dimensional (e.g., static or dynamic suspensioncultures) culture systems. Thus, hESCs or human iPS cells culturedaccording to the teachings of the present invention can be used as asource for generating differentiated, lineage-specific cells. Such cellscan be obtained directly from the ESCs by subjecting the ESCs to variousdifferentiation signals (e.g., cytokines, hormones, growth factors) orindirectly, via the formation of embryoid bodies and the subsequentdifferentiation of cells of the EBs to lineage-specific cells.

Thus, according to an aspect of the some embodiments of the inventionthere is provided a method of generating embryoid bodies frompluripotent stem cells. The method is effected by (a) culturing thepluripotent stem cells according to the method of some embodiment of theinvention to thereby obtain expanded, undifferentiated pluripotent stemcells; and (b) subjecting the expanded, undifferentiated pluripotentstem cells to culturing conditions suitable for differentiating the stemcells to embryoid bodies, thereby generating the embryoid bodies fromthe pluripotent stem cells.

As used herein the phrase “embryoid bodies” refers to morphologicalstructures comprised of a population of ESCs, extended blastocyst cells(EBCs), embryonic germ cells (EGCs) and/or induced pluripotent stemcells which have undergone differentiation. EBs formation initiatesfollowing the removal of differentiation blocking factors from thepluripotent stem cell cultures. In the first step of EBs formation, thepluripotent stem cells proliferate into small masses of cells which thenproceed with differentiation. In the first phase of differentiation,following 1-4 days in culture for either human ESCs or human iPS cells,a layer of endodermal cells is formed on the outer layer of the smallmass, resulting in “simple EBs”. In the second phase, following 3-20days post-differentiation, “complex EBs” are formed. Complex EBs arecharacterized by extensive differentiation of ectodermal and mesodermalcells and derivative tissues.

Thus, the method according to some embodiments of the invention involvesthe culturing of the pluripotent stem cells in any of the culture mediadescribed hereinabove in order to obtain expanded, undifferentiatedpluripotent stem cells and then subjecting the expanded,undifferentiated pluripotent stem cells (e.g., ESCs or iPS cells) toculturing conditions suitable for differentiating the pluripotent stemcells to embryoid bodies. Such culturing conditions are substantiallydevoid of differentiation inhibitory factors which are employed whenpluripotent stem cells are to be expanded in an undifferentiated state,such as TGFβ₃, ascorbic acid at a concentration of at least 50 μg/ml,bFGF and/or the IL6RIL6 chimera.

For EBs formation, the pluripotent stem cells (ESCs or iPS cells) areremoved from their feeder cell layers, feeder-free-culturing systems orsuspension cultures and are transferred to a suspension culture in thepresence of a culture medium containing serum or serum replacement andbeing devoid of differentiation-inhibitory factors (see e.g., Examples1, 2, 4 and 5 of the Examples section which follows). For example, aculture medium suitable for EBs formation may include a basic culturemedium (e.g., Ko-DMEM or DMEM/F12) supplemented with 20% FBSd (HyClone,Utah, USA). 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, and 1%non-essential amino acid stock.

Monitoring the formation of EBs is within the capabilities of thoseskilled in the art and can be effected by morphological evaluations(e.g., histological staining) and determination of expression ofdifferentiation-specific markers [e.g., using immunological techniquesor RNA-based analysis (e.g., RT-PCR, cDNA microarray)].

It will be appreciated that in order to obtain lineage-specific cellsfrom the EBs, cells of the EBs can be further subjected to culturingconditions suitable for lineage-specific cells.

Preferably, the method of this aspect of the present invention furtherincludes step (c) of subjecting cells of the embryoid bodies toculturing conditions suitable for differentiating and/or expandinglineage specific cells; thereby generating the lineage-specific cellsfrom the embryonic stem cells.

As used herein the phrase “culturing conditions suitable fordifferentiating and/or expanding lineage specific cells” refers to acombination of culture system, e.g., feeder cell layers, feeder-freematrix or a suspension culture and a culture medium which are suitablefor the differentiation and/or expansion of specific cell lineagesderived from cells of the EBs. Non-limiting examples of such culturingconditions are further described hereinunder.

According to some embodiments of the invention, the method of thisaspect of the invention further includes isolating lineage specificcells following step (b).

As used herein, the phrase “isolating lineage specific cells” refers tothe enrichment of a mixed population of cells in a culture with cellspredominantly displaying at least one characteristic associated with aspecific lineage phenotype. It will be appreciated that all celllineages are derived from the three embryonic germ layers. Thus, forexample, hepatocytes and pancreatic cells are derived from the embryonicendoderm, osseous, cartilaginous, elastic, fibrous connective tissues,myocytes, myocardial cells, bone marrow cells, vascular cells (namelyendothelial and smooth muscle cells), and hematopoietic cells aredifferentiated from embryonic mesoderm and neural, retina and epidermalcells are derived from the embryonic ectoderm.

According to some preferred embodiments of the invention, isolatinglineage specific cells is effected by sorting of cells of the EBs viafluorescence activated cell sorter (FACS).

Methods of isolating EB-derived-differentiated cells via FACS analysisare known in the art. According to one method, EBs are disaggregatedusing a solution of Trypsin and EDTA (0.025% and 0.01%, respectively),washed with 5% fetal bovine serum (FBS) in phosphate buffered saline(PBS) and incubated for 30 min on ice with fluorescently-labeledantibodies directed against cell surface antigens characteristics to aspecific cell lineage. For example, endothelial cells are isolated byattaching an antibody directed against the platelet endothelial celladhesion molecule-1 (PECAM1) such as the fluorescently-labeled PECAM1antibodies (30884X) available from PharMingen (PharMingen, BectonDickinson Bio Sciences, San Jose, Calif., USA) as described inLevenberg, S. et al., (Endothelial cells derived from human embryonicstem cells. Proc. Natl. Acad. Sci. USA. 2002. 99: 4391-4396).Hematopoietic cells are isolated using fluorescently-labeled antibodiessuch as CD34-FITC, CD45-PE, CD31-PE, CD38-PE, CD90-FITC, CD117-PE,CD15-FITC, class I-FITC, all of which IgG1 are available fromPharMingen. CD133/1-PE (IgG1) (available from Miltenyi Biotec, Auburn,Calif.), and glycophorin A-PE (IgG1), available from Immunotech (Miami,Fla.). Live cells (i.e., without fixation) are analyzed on a FACScan(Becton Dickinson Bio Sciences) by using propidium iodide to excludedead cells with either the PC-LYSIS or the CELLQUEST software. It willbe appreciated that isolated cells can be further enriched usingmagnetically-labeled second antibodies and magnetic separation columns(MACS, Miltenyi) as described by Kaufman. D. S. et al., (Hematopoieticcolony-forming cells derived from human embryonic stem cells. Proc.Natl. Acad. Sci. USA. 2001, 98: 10716-10721).

According to some embodiments of the invention, isolating lineagespecific cells is effected by a mechanical separation of cells, tissuesand/or tissue-like structures contained within the EBs.

For example, beating cardiomyocytes can be isolated from EBs asdisclosed in U.S. Pat. Appl. No. 20030022367 to Xu et al. Four-day-oldEBs of the present invention are transferred to gelatin-coated plates orchamber slides and are allowed to attach and differentiate.Spontaneously contracting cells, which are observed from day 8 ofdifferentiation, are mechanically separated and collected into a 15-mLtube containing low-calcium medium or PBS. Cells are dissociated usingCollagenase B digestion for 60-120 minutes at 37° C., depending on theCollagenase activity. Dissociated cells are then resuspended in adifferentiation KB medium (85 mM KCl. 30 mM K₂HPO₄, 5 mM MgSO₄, 1 mMEGTA. 5 mM creatine, 20 mM glucose. 2 mM Na₂ATP, 5 mM pyruvate, and 20mM taurine, buffered to pH 7.2. Maltsev et al., Circ. Res. 75:233, 1994)and incubated at 37° C. for 15-30 min. Following dissociation cells areseeded into chamber slides and cultured in the differentiation medium togenerate single cardiomyocytes capable of beating.

According to some embodiments of the invention, isolating lineagespecific cells is effected by subjecting the EBs to differentiationfactors to thereby induce differentiation of the EBs into lineagespecific differentiated cells.

Following is a non-limiting description of a number of procedures andapproaches for inducing differentiation of EBs to lineage specificcells.

To differentiate the EBs of some embodiments of the invention intoneural precursors, four-day-old EBs are cultured for 5-12 days in tissueculture dishes including DMEM/F-12 medium with 5 mg/ml insulin. 50 mg/mltransferrin, 30 nM selenium chloride, and 5 mg/ml fibronectin (ITSFnmedium. Okabe, S. et al., 1996, Mech. Dev. 59: 89-102). The resultantneural precursors can be further transplanted to generate neural cellsin vivo (Brüstle, O. et al., 1997. In vitro-generated neural precursorsparticipate in mammalian brain development. Proc. Natl. Acad. Sci. USA.94: 14809-14814). It will be appreciated that prior to theirtransplantation, the neural precursors are trypsinized and triturated tosingle-cell suspensions in the presence of 0.1% DNase.

EBs of some embodiments of the invention can differentiate tooligodendrocytes and myelinate cells by culturing the cells in modifiedSATO medium. i.e., DMEM with bovine serum albumin (BSA), pyruvate,progesterone, putrescine, thyroxine, triiodothryonine, insulin,transferrin, sodium selenite, amino acids, neurotrophin 3, ciliaryneurotrophic factor and Hepes (Bottenstein. J. E. & Sato, G. H., 1979.Proc. Natl. Acad. Sci. USA 76, 514-517; Raff, M. C., Miller, R. H., &Noble, M., 1983. Nature 303: 390-396]. Briefly, EBs are dissociatedusing 0.25% Trypsin/EDTA (5 min at 37° C.) and triturated to single cellsuspensions. Suspended cells are plated in flasks containing SATO mediumsupplemented with 5% equine serum and 5% fetal calf serum (FCS).Following 4 days in culture, the flasks are gently shaken to suspendloosely adhering cells (primarily oligodendrocytes), while astrocytesare remained adhering to the flasks and further producing conditionedmedium. Primary oligodendrocytes are transferred to new flaskscontaining SATO medium for additional two days. Following a total of 6days in culture, oligospheres are either partially dissociated andresuspended in SATO medium for cell transplantation, or completelydissociated and a plated in an oligosphere-conditioned medium which isderived from the previous shaking step [Liu, S. et al., (2000).Embryonic stem cells differentiate into oligodendrocytes and myelinatein culture and after spinal cord transplantation. Proc. Natl. Acad. Sci.USA. 97: 6126-6131].

For mast cell differentiation, two-week-old EBs of some embodiments ofthe invention are transferred to tissue culture dishes including DMEMmedium supplemented with 10% FCS, 2 mM L-glutamine, 100 units/mlpenicillin, 100 mg/ml streptomycin, 20% (v/v) WEHI-3 cell-conditionedmedium and 50 ng/ml recombinant rat stem cell factor (rrSCF, Tsai, M. etal., 2000. In vivo immunological function of mast cells derived fromembryonic stem cells: An approach for the rapid analysis of evenembryonic lethal mutations in adult mice in vivo. Proc Natl Acad SciUSA. 97: 9186-9190). Cultures are expanded weekly by transferring thecells to new flasks and replacing half of the culture medium.

To generate hemato-lymphoid cells from the EBs of some embodiments ofthe invention. 2-3 days-old EBs are transferred to gas-permeable culturedishes in the presence of 7.5% CO₂ and 5% O₂ using an incubator withadjustable oxygen content. Following 15 days of differentiation, cellsare harvested and dissociated by gentle digestion with Collagenase (0.1unit/mg) and Dispase (0.8 unit/mg), both are available from F.Hoffman-La Roche Ltd, Basel, Switzerland. CD45-positive cells areisolated using anti-CD45 monoclonal antibody (mAb) M1/9.3.4.HL.2 andparamagnetic microbeads (Miltenyi) conjugated to goat anti-ratimmunoglobulin as described in Potocnik, A. J. et al., (ImmunologyHemato-lymphoid in vivo reconstitution potential of subpopulationsderived from in vitro differentiated embryonic stem cells. Proc. Natl.Acad. Sci. USA. 1997, 94: 10295-10300). The isolated CD45-positive cellscan be further enriched using a single passage over a MACS column(Miltenyi).

It will be appreciated that the culturing conditions suitable for thedifferentiation and expansion of the isolated lineage specific cellsinclude various tissue culture media, growth factors, antibiotic, aminoacids and the like and it is within the capability of one skilled in theart to determine which conditions should be applied in order to expandand differentiate particular cell types and/or cell lineages.

Additionally or alternatively, lineage specific cells can be obtained bydirectly inducing the expanded, undifferentiated pluripotent stem cellssuch as ESCs or iPS cells to culturing conditions suitable for thedifferentiation of specific cell lineage.

According to an aspect of some embodiments of the invention there isprovided a method of generating lineage-specific cells from pluripotentstem cells. The method is effected by (a) culturing the pluripotent stemcells according to the method of some embodiments of the invention, tothereby obtain expanded, undifferentiated stem cells; and (b) subjectingthe expanded, undifferentiated stem cells to culturing conditionssuitable for differentiating and/or expanding lineage specific cells,thereby generating the lineage-specific cells from the pluripotent stemcells.

Following are non-limiting examples of culturing conditions which aresuitable for differentiating and/or expanding lineage specific cellsfrom pluripotent stem cells (e.g., ESCs and iPS cells).

Mesenchymal stromal cells which are CD73-positive and SSEA-4-negativecan be generated from hESCs by mechanically increasing the fraction offibroblast-like differentiated cells formed in cultures of hESCs,essentially as described in Trivedi P and Hematti P. Exp Hematol. 2008,36(3):350-9. Briefly, to induce differentiation of hESC the intervalsbetween medium changes are increased to 3-5 days, and the cells at theperiphery of the ESC colonies become spindle-shaped fibroblast-lookingcells. After 9-10 days under these conditions when about 40-50% of thecells in the culture acquire the fibroblast-looking appearance, theundifferentiated portions of ESC colonies are physically removed and theremaining differentiated cells are passaged to new culture plates underthe same conditions.

To induce differentiation of hESCs into dopaminergic (DA) neurons, thecells can be co-cultured with the mouse stromal cell lines PA6 or MS5,or can be cultured with a combination of stromal cell-derived factor 1(SDF-1/CXCL12), pleiotrophin (PTN), insulin-like growth factor 2 (IGF2)and ephrin BI (EFNB1) essentially as described in Vazin T, et al., PLoSOne. 2009 Aug. 12; 4(8):e6606; and in Elkabetz Y., et al., Genes Dev.2008 Jan. 15; 22: 152-165.

To generate mesencephalic dopamine (mesDA) neurons, hESCs can begenetically modified to express the transcription factor Lmx1a (e.g.,using a lentiviral vector with the PGK promoter and Lmx1a) essentiallyas described in Friling S., et al., Proc Natl Acad Sci USA. 2009, 106:7613-7618.

To generate lung epithelium (type I pneumocytes) from hESCs, the ESCscan be cultured in the presence of a commercially available cell culturemedium (Small Airway Growth Medium; Cambrex, College Park, Md.), oralternatively, in the presence of a conditioned medium collected from apneumocyte cell line (e.g., the A549 human lung adenocarcinoma cellline) as described in Rippon H J., et al., Proc Am Thorac Soc.2008:5:717-722.

To induce differentiation of hESCs or human iPS cells into neural cells,the pluripotent stem cells can be cultured for about 5 days in thepresence of a serum replacement medium supplemented with TGF-b inhibitor(SB431542, Tocris; e.g., 10 nM) and Noggin (R&D; e.g., 500 ng/ml),following which the cells are cultured with increasing amounts (e.g.,25%, 50%, 75%, changed every two days) of N2 medium (Li X J., et al.,Nat Biotechnol. 2005, 23:215-21) in the presence of 500 ng/mL Noggin,essentially as described in Chambers S M., et al., Nat Biotechnol. 2009,27: 275-280.

In addition to the lineage-specific primary cultures, EBs of theinvention can be used to generate lineage-specific cell lines which arecapable of unlimited expansion in culture.

Cell lines of the present invention can be produced by immortalizing theEB-derived cells by methods known in the art, including, for example,expressing a telomerase gene in the cells (Wei, W. et al., 2003. MolCell Biol. 23:2859-2870) or co-culturing the cells with NIH 3T3hph-HOX11 retroviral producer cells (Hawley, R. G. et al., 1994.Oncogene 9: 1-12).

It will be appreciated that since the lineage-specific cells or celllines obtained according to the teachings of the invention are developedby differentiation processes similar to those naturally occurring in thehuman embryo they can be further used for human cell-based therapy andtissue regeneration.

Thus, the invention envisages the use of the expanded and/ordifferentiated lineage-specific cells or cell lines of some embodimentsof the invention for treating a disorder requiring cell replacementtherapy.

For example, oligodendrocyte precursors can be used to treat myelindisorders (Repair of myelin disease: Strategies and progress in animalmodels. Molecular Medicine Today. 1997. pp. 554-561), chondrocytes ormesenchymal cells can be used in treatment of bone and cartilage defects(U.S. Pat. No. 4,642,120) and cells of the epithelial lineage can beused in skin regeneration of a wound or burn (U.S. Pat. No. 5,716,411).

For certain disorders, such as genetic disorders in which a specificgene product is missing [e.g., lack of the CFTR gene-product in cysticfibrosis patients (Davies J C, 2002. New therapeutic approaches forcystic fibrosis lung disease. J. R. Soc. Med. 95 Suppl 41:58-67)],ESC-derived cells or iPS cells-derived cells are preferably manipulatedto over-express the mutated gene prior to their administration to theindividual. It will be appreciated that for other disorders, theESC-derived cells or iPS-derived cells should be manipulated to excludecertain genes.

Over-expression or exclusion of genes can be effected using knock-inand/or knock-out constructs [see for example, Fukushige, S. and Ikeda.J. E.: Trapping of mammalian promoters by Cre-lox site-specificrecombination. DNA Res 3 (1996) 73-50; Bedell, M. A., Jerkins, N. A. andCopeland, N. G.: Mouse models of human disease. Part I: Techniques andresources for genetic analysis in mice. Genes and Development 11 (1997)1-11; Bermingham. J. J., Scherer, S. S., O'Connell, S., Arroyo. E.,Kalla, K. A., Powell, F. L. and Rosenfeld. M. G.: Tst-1/Oct-6/SCIPregulates a unique step in peripheral myelination and is required fornormal respiration. Genes Dev 10 (1996) 1751-62].

In addition to cell replacement therapy, the lineage specific cells ofsome embodiments of the invention can also be utilized to prepare a cDNAlibrary. mRNA is prepared by standard techniques from the lineagespecific cells and is further reverse transcribed to form cDNA. The cDNApreparation can be subtracted with nucleotides from embryonicfibroblasts and other cells of undesired specificity, to produce asubtracted cDNA library by techniques known in the art.

The lineage specific cells of some embodiments of the invention can beused to screen for factors (such as small molecule drugs, peptides,polynucleotides, and the like) or conditions (such as culture conditionsor manipulation) that affect the differentiation of lineage precursor toterminally differentiated cells. For example, growth affectingsubstances, toxins or potential differentiation factors can be tested bytheir addition to the culture medium.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”. “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 12, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”. Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4. ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange. Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait. M. J., ed. (1984); “Nucleic AcidHybridization” Hames. B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney. R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal. B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”.Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996) all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader.

All the information contained therein is incorporated herein byreference.

General Materials and Experimental Methods

Cell Lines

iPS cell culture—Induced pluripotent stem (iPS) cell lines J1.2-3 andiF4 [Park et al, 2008] derived from foreskin fibroblast and adultfibroblasts respectively, were cultured with inactivated mouse embryonicfibroblasts (MEF) as was previously described [Park et al, 2008]. Thefollowing culture media combinations were tested for their ability tosupport the growth of iPS cells in attached [two-dimensional (2D)]cultures:

hESC culture—The human ESC lines I4, I3, I6 and H9.2 were used in thestudy.

Culturing conditions on two-dimensions: hESCs lines or human iPS celllines were cultured with MEFs or on synthetic matrices in the presenceof the tested culture media. Cells were passaged every four to six daysusing 1 mg/ml type IV collagenase (Gibco Invitrogen Corporation. GrandIsland N.Y., USA) and plated at a density of 1×10⁴-3×10⁵ cells per cm².

Media Used for 2D Cultures—

(i) yF10 basic culture medium consisting of 85% DMEM/F12 (BiologicalIndustries, Beit Haemek, Israel), 15% KNOCKOUT™ serum replacement (SR;Invitrogen), 2 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1%non-essential amino acid stock, and 10 ng/ml basic fibroblast growthfactor (bFGF), all from Invitrogen Corporation products, Grand IslandN.Y., USA, unless otherwise indicated. This basic culture medium wasused as a control and for the routine growth of iPS cells or hESCs withinactivated MEF or foreskin fibroblasts as feeder layers in 2D cultures.

(ii) mHA40/4 DMEM/F12 (94%) (Biological Industries, Israel), ITS 1%[Invitrogen corporation; the ITS premix is a ×100 stock solutionconsists of 1.25 mg Insulin, 1.25 mg Transferrin and 1.25 mg Seleniusacid], 2 ng/ml TGFβ₃ (from R&D Systems Minneapolis Minn. USA).L-glutamine 2 mM (Invitrogen corporation), ascorbic acid 500 μg/ml(Sigma, Israel), bFGF—10 ng (Invitrogen corporation), Human serumalbumin—0.5% (Sigma, Catalogue No. A1653). Na-Bicarbonate (7.5%)(Biological Industries. Israel), defined lipid mixture 1% (Invitrogencorporation).

(iii) HA75 DMEM/F12 (94%) (Biological Industries, Israel). L-glutamine 2mM (Invitrogen corporation), ascorbic acid 500 μg/ml (Sigma), bFGF—10 ng(Invitrogen Corporation). TGFβ₃ 2 ng/ml (R&D Systems Minneapolis Minn.USA). SR3 (serum replacement)—1% (Sigma, Israel), defined lipid mixture1% (Invitrogen corporation).

(iv) HA76 DMEM/F12 (94%) (Biological Industries. Beit HaEmek, Israel).ITS 1% (Invitrogen corporation), L-glutamine 2 mM (Invitrogencorporation), ascorbic acid 500 μg/ml (Sigma, Israel), bFGF—100 ng(Invitrogen corporation). TGFβ₃ 2 ng/ml (R&D Systems Minneapolis Minn.,USA). Human serum albumin serum—1% (Sigma, Catalogue No. A1653).Na-Bicarbonate (7.5%) (Biological Industries, Israel), defined lipidmixture 1% (Invitrogen corporation).

(v) HA77 DMEM/F12 (94%) (Biological Industries. Israel, Sigma Israel),L-glutamine 2 mM (Invitrogen corporation, Sigma, Israel), ascorbic acid500 μg/ml (Sigma, Israel), bFGF—100 ng (Invitrogen corporation),Na-Bicarbonate (7.5%) (Biological Industries, Israel). SR3—1% (Sigma,Israel), defined lipid mixture 1% (Invitrogen corporation, Sigma,Israel). It should be noted that the HA77 DMEM/F12 (94%) can also beused without Na-Bicarbonate at all and yet support the culture ofpluripotent stem cells (e.g., hESCs and iPSCs) in a proliferative,pluripotent and undifferentiated state for at least 10 passages.

(vi) HA78 DMEM/F12 (94%) (Biological Industries, Israel), L-glutamine 2mM (Invitrogen corporation), ascorbic acid 500 μg/ml (Sigma, Israel),bFGF—10 ng/ml (Invitrogen corporation), TGFβ₃ 2 ng/ml (R&D SystemsMinneapolis Minn., USA). SR3™—1% (Sigma, Israel), Na-Bicarbonate (7.5%)(Biological Industries, Israel), defined lipid mixture 1% (Invitrogencorporation).

(v) HA74/1 DMEM/F12 (94%) (Biological Industries, Israel). ITS 1%(Invitrogen corporation). L-glutamine 2 mM (Invitrogen corporation),ascorbic acid 500 μg/ml (Sigma, Israel), bFGF—50 ng/ml (Invitrogencorporation), TGFβ₃ 2 ng/ml (R&D Systems Minneapolis Minn. USA), Humanserum albumin—0.5% (Sigma, Israel, Catalogue No. A1653), Na-Bicarbonate(7.5%) (Biological Industries, Israel), defined lipid mixture 1%(Invitrogen Corporation).

It should be noted that when recombinant human Albumin (SIGMA. CatalogueNo. A7223) was used instead of human serum Albumin (SIGMA, Catalogue No.A1653) in the mHA40/4, HA76. HA74/1 culture media, these culture mediawere found to support the growth of hESCs and iPS cells in a pluripotentand undifferentiated state for an extended period of culture. Thus,these results demonstrate that recombinant human albumin can be usedinstead of human serum albumin in the culture media of some embodimentsof the invention and thereby provide defined, xeno-free conditions.

Culturing Conditions in Three-Dimension Culture Systems (SuspensionCulture):

Media Used for Suspension Cultures—

(i) CM100Fp medium consisting of the basic culture medium (yF10 basicculture medium) supplemented with 100 μg/ml IL6RIL6 chimera. The 85-KdaIL6RIL6 was produced and purified as described and was donated byInterPharm, Merck-Serono group (Nes-Ziona, Israel and Geneva,Switzerland).

(ii) CM100F medium consisting of the basic culture medium (yF10 basicculture medium) supplemented with 100 ng/ml IL6RIL6 chimera. The 85-KdaIL6RIL6 was produced and purified as described and was donated byInterPharm. Merck-Serono group (Nes-Ziona, Israel and Geneva,Switzerland).

(iii) yF100 basic medium (yF10 basic culture medium) in which instead of10 ng/ml bFGF 100 ng/ml bFGF was used. This medium was found to supporthESCs suspension culture with the same efficiency as CM100F.

(iv) yFL3 medium consists of the yF10 basic culture medium with 4 ng/mlbFGF instead of 10 ng/ml bFGF, and supplemented with 3000 units/mlleukemia inhibitory factor (LIF). It should be noted that iPS cells werecultured with the yFL3 medium which comprised 4 or 10 ng/ml bFGF withthe same efficiency.

(v) modified HA13(a) medium consists of DMEM/F12 (95%). L-glutamine 2mM, ascorbic acid 500 μg/ml, bFGF—4 ng, and SR3—1% J. Was found tosupport hESCs and iPSCs in a 2-dimensional and 3-dimensional culturesystems.

(vi) modified HA13(b) medium consists of DMEM/F12 (95%), L-glutamine 2mM, ascorbic acid 500 μg/ml, bFGF—4 ng, SR3—1% and a lipid mixture(1%)]. Was found to support hESCs and iPSCs in a 2-dimensional and3-dimensional culture systems.

(vii) modified HA13(c) medium consists of DMEM/F12 (95%). L-glutamine 2mM, ascorbic acid 50 μg/ml, bFGF—4 ng, and SR3—1%. Was found to supporthESCs and iPSCs in a 2-dimensional and 3-dimensional culture systems.

(viii) modified HA13(d) medium consists of DMEM/F12 (95%), L-glutamine 2mM, ascorbic acid 50 μg/ml, bFGF—4 ng, SR3—1% and a lipid mixture (1%)].Was found to support hESCs and iPSCs in a 2-dimensional and3-dimensional culture systems.

Culture in static suspension (3-dimensional) cultures—To initiatesuspension cultures, the iPS cells or the hESCs were removed from theirculture dish using 1.5 mg/ml type IV collagenase (Worthingtonbiochemical corporation, Lakewood, N.J. USA), further broken into smallclumps using 200 μl Gilson pipette tips, and cultured in suspension in58 mm Petri dishes (Greiner, Frickenhausen, Germany) at a cell densityof 1×10⁶-5×10⁶ cells/dish (5-8 ml of medium in 58-mm dishes). The Petridishes were kept static in an incubator at 37° C. in 5% CO₂. Whenrequired, differentiating clumps were removed from the culture duringthe first three passages while the cells adapted to the new cultureconditions. The medium in the suspension culture was changed daily, andthe cells were passaged every 5-7 days either by manual cutting ofclumps using 27 g needles (only at passages 1-3) or by gentle pipettingusing 200 ml Gilson pipette tips. Alternatively, the cells were passagedusing trypsin EDTA (0.25%, Biological Industries, Beit Haemek, Israel)combined with a one-hour treatment with 10 M ROCK inhibitor (EMDBiosciences, Inc. La Jolla, Calif. USA) before the incubation withtrypsin. For calculating cells' doubling time the I3, I4 and H9.2 hESCsand the J1.2-3 and iF4 iPS cells were counted and grown in suspensionfor 8 days with CM100F or CM100Fp culture media. Cells were countedevery other day. Average doubling time of four biological repeats wascalculated.

Culture in spinner flasks (3-dimensional)—iPS cells or hESCs clumps werecultured in static Petri dishes for at least one passage, and were thentransferred to 250 ml spinner flasks (Cell Spin 250 or 100, IntegraBioSciences) in the tested culture media, shaken continuously at 90rounds per minutes (rpm) using a magnetic plate, and placed in a 37° C.in 5% CO₂ incubator. Medium was changed every other day. Every 5-7 daysthe clumps were split with a ratio of 1:2.

Immunofluorescence of cells cultured on 2-D or 3-D culture systems—Forfluorescent immunostaining undifferentiated hESCs or iPS grown in 2-D or3-D culture systems in the presence of the tested culture media orre-cultured on MEFs were fixed with 4% paraformaldehyde and exposed tothe primary antibodies overnight at 4° C. Cys 3 conjugated antibodies(Chemicon International, Temecula Calif., USA) were used as secondaryantibodies (1:200). The primary antibodies (1:50) include SSEA 1, 3 and4 (Hybridoma Bank. Iowa, USA), TRA1-60 and TRA-81 (ChemiconInternational, Temecula Calif., USA) and Oct4 (Santa Cruz Biotechnology.Santa Cruz, Calif., USA).

Immunohistochemistry of iPS cells or hESCs cultured on 2-D or 3-Dculture systems—After deparaffinization the tissue sections were stainedusing Dako LSAB+ staining kit for presence of markers of ectoderm(β-3-tubulin 1:500, Chemicon International, Temecula Calif. USA),mesoderm (CD31 1:20), and endoderm (α-fetoprotein 1:20) (both fromDakoCytomation, Glostrup, Denmark). As controls, both IgG isotype andsecondary antibody staining were performed. The secondary antibody wasconjugated to peroxidase.

Karyotype analysis of cells cultured on 2-D or 3-D culturesystems—Karyotype analysis (G-banding) was performed on at least 10cells from each sample, two samples per test, as previously described[Amit et al. 2003]. Karyotypes were analyzed and reported according tothe “International System for Human Cytogenetic Nomenclature” (ISCN).

Embryoid bodies (EBs) formation of cells cultured on 2-D or 3-D culturesystems—For the formation of EBs, hESCs or iPS were passaged asdescribed and transferred to 58 mm Petri dishes (Greiner, Frickenhausen,Germany). EBs were grown in medium consisting of 80% DMEM/F12(Biological Industries. Beit Haemek, Israel), supplemented with 10%fetal bovine serum (FBS) (HyClone, Utah, USA), 10% KNOCKOUT™ serumreplacement (SR; Invitrogen), 2 mM L-glutamine, 0.1 mMβ-mercaptoethanol, and 1% non-essential amino acid stock (InvitrogenCorporation, Grand Island N.Y. USA). 10-14 day-old EBs were harvestedfor RNA isolation and histological examination. For histologicalanalysis EBs were fixed in 10% neutral-buffered formalin, dehydrated ingraduated alcohol (70%-100%) and embedded in paraffin. □ 1-5 μm sectionswere deparaffinized and stained with hematoxylin/eosin (H&E).

RT-PCR of cells cultured on 2-D or 3-D culture systems—Total RNA wasisolated from hESCs or iPS grown for 10, 15 and 20 passages on thexeno-free two-dimensional or three-dimensional culture systems in thetested media and from 10-14 day-old EBs (formed from cells grown on 2-D,3-D in the presence of the tested culture media or cells cultured onMEFs) using Tri-Reagent (Sigma, St. Louis Mo., USA), according to themanufacturer's instructions. cDNA was synthesized from 1 μg total RNAusing MMLV reverse transcriptase RNase H minus (Promega, Madison Wis.USA). PCR reaction included denaturation for 5 minutes at 94° C.followed by repeated cycles of 94° C. for 30 seconds, annealingtemperature as indicated in Table 1, hereinbelow, for 30 seconds andextension at 72° C. for 30 seconds. PCR primers and reaction conditionsare described in Table 1, hereinbelow. PCR products weresize-fractionated using 2% agarose gel electrophoresis. DNA markers wereused to confirm the size of the resultant fragments. For quantitativePCR (Q-PCR), densitometry of tested genes was normalized to GAPDH. Threerepeats were conducted for each tested line.

TABLE 1 RT-PCR primers and conditionsTable 1: RT-PCR primers and PCR conditions are provided along with the GenBank Accession numbers of the amplified transcripts.Gene product Forward (F) (Accession and reverse (R) primers (SEQ ID NO:)Reaction Size number) provided in a 5′→3′ direction Condition (bp) Oct-4F: 5′-GAGAACAATGAGAACCTTCAGGA (SEQ ID 30 cycles 219 (S81255) NO: 1.)at 60° C. R: 5′-TTCTGCGCCGGTTACAGAACCA (SEQ ID in 1.5 mM NO: 2) MgCl₂Albumin F: 5′-TGCTTGAATCAGCTGATGACAGGG (SEQ ID 35 cycles 302 (AF542069)NO: 3) at 60° C. R: 5′-AAGGCAAGTCAGCAGCCATGTCAT (SEQ ID in 1.5 mM NO: 4)MgCl₂ α-fetoprotein F: 5′-GCTGGATTCTTCTGCAGGATGGGGAA (SEQ ID 30 cycles216 (BC027881) NO: 5) at 60° C. R: 5′-TCCCCTGAAGAAAATTGGTTAAAAT (SEQ IDin 1.5 mM NO: 6) MgCl₂ NF-68KDF: 5′-GAGTGAAATGGCACGATACCTA (SEQ ID NO: 7) 30 cycles 473 (NFHR: 5′-TTTCCTCTCCTTCTTCACCTTC (SEQ ID NO: 8) at 60° C. (AY156690; in 2 mMX15307; MgCl₂ X15309) α-cardiacF: 5′-GGAGTTATGGTGGGTATGGGTC (SEQ ID NO: 9) 35 cycles 486 actinR: 5′-AGTGGTGACAAAGGAGTAGCCA (SEQ ID at 65° C. (NM_005159) NO: 10)in 2 mM MgCl₂ β-Actin F: 5′-ATCTGGCACCACACCTTCTACAATGAGCTGCG 35 cycles838 (NM_001101) (SEQ ID NO: 11) at 62° C.R: 5′-CGTCATACTCCTGCTTGCTGATCCACATCTGC in 1.5 mM (SEQ ID NO: 12) MgCl₂Sox2 F: 5′CCCCCGGCGGCAATAGCA (SEQ ID NO: 13) 35 cycles 448 (Z31560)R: 5′TCGGCGCCGGGGAGATACAT (SEQ ID NO: 14) at 60° C. in 1.5 mM MgC1₂ Rex1F: 5′GCGTACGCAAATTAAAGTCCAGA (SEQ ID 35 cycles 306 (AF450454) NO: 15)at 56° C. R: 5′CAGCATCCTAAACAGCTCGCAGAAT (SEQ ID in 1.5 mM NO: 16) MgCl₂CX43 F: 5′TACCATGCGACCAGTGGTGCGCT (SEQ ID 35 cycles 295 (NM_000165)NO: 17) at 61° C. R: 5′GAATTCTGGTTATCATCGGGGAA (SEQ ID in 1.5 mM NO: 18)MgCl₂ FGE4 F: 5′CTACAACGCCTACGAGTCCTACA (SEQ ID 35 cycles 370(NM_002007) NO: 19) at 52° C. R: 5′GTTGCACCAGAAAAGTCAGAGTTG (SEQ IDin 1.5 mM NO: 20) MgCl₂ Glucagon F: 5′CTCAGTGATCCTGATCAGATGAACG (SEQ ID35 cycles 370 (X03991) NO: 21) at 65° C.R: 5′AGTCCCTGGCGGCAAGATTATCAAG (SEQ ID in 1.5 mM NO: 22) MgC1₂β-globulin F: 5′ACCTGACTCCTGAGGAGAAGTCTGC (SEQ ID 35 cycles 410 (V00499)NO: 23) at 65° C. R: 5′TAGCCACACCAGCCACCACTTTCTG (SEQ ID in 1.5 mMNO: 24) MgC1₂ Flk1 F: 5′ATGCACGGCATCTGGGAATC (SEQ ID NO: 25) 35 cycles537 (NM_002253) R: 5′GCTACTGTCCTGCAAGTTGCTGTC (SEQ ID at 65° C. NO: 26)in 1.5 mM MgCl₂ AC133 F: 5′CAGTCTGACCAGCGTGAAAA (SEQ ID NO: 27)35 cycles 200 (NM_006017) R: 5′GGCCATCCAAATCTGTCCTA (SEQ ID NO: 28)at 65° C. in 1.5 mM MgCl₂ NanogF: 5′ACTAACATGAGTGTGGATCC (SEQ ID NO: 29) 35 cycles 800 (NG_004095)R: 5′TCATCTTCACACGTCTTCAG (SEQ ID NO :30) at 61° C. in 1.5 mM MgCl₂

Teratoma formation from cells cultured on 2-D—hESCs (H9.2 and 13) andiPS (iF4 and J1.2-3) cells from 4-6 wells of a 6-well plate (each wellhas 10 cm total surface area and includes 1.5-2.5×10⁶ cells) wereharvested and injected into the hindlimb muscles of four week-old maleof severe combined immunodeficiency (SCID)-beige mice. Ten weeks afterthe injection the resultant teratomas were harvested and prepared forhistological analysis using the same method mentioned for EBs.

Teratoma formation from cells cultured in suspension (3-D culturesystems)—hESCs (H9.2 and 13) and iPS (iF4 and J1.2-3) cells from four tosix 58 mm dishes (from suspension culture, each dish includes1.5-2.5×10⁶ cells) were harvested and injected into the hindlimb musclesof four week-old male of severe combined immunodeficiency (SCID)-beigemice. Ten weeks after the injection the resultant teratomas wereharvested and prepared for histological analysis using the same methodmentioned for EBs.

Example 1 Induced Pluripotent Stem Cells and Embryonic Stem Cells can beMaintained in an Undifferentiated and Pluripotent State when Cultured onXeno-Free, Feeder-Layer-Free 2-D Culture Systems

The experiments described hereinbelow were performed using iPS cells orhESCs which were cultured according to the methods, culturing conditionsand culture media described in the “General Materials and ExperimentalMethods” section above.

Experimental Results

iPS cells and human ESCs cultured on 2D culture systems using xeno-free,serum-free medium and supportive-layers free system exhibitundifferentiated morphology and characteristics typical to iPS orhESCs—Several possible medium combinations (HA74/1, HA75. HA76. HA77.HA78. HA40\4) were tested for the ability to support feeder-layer freeand xeno-free (devoid of any animal contaminant) cultures of iPS cellsor hESCs. All tested media (i.e., HA74/1, HA75. HA76. HA77. HA78,HA40\4) were found suitable for supporting iPS or hESC cultures for atleast 15 passages. Using the tested media under feeder-layer freeconditions using a Matrigel™ synthetic matrix iPS cells or hESCs werecultured continuously for at least 15 passages while maintaining theiriPS of hESCs features including undifferentiated proliferation,karyotype stability and pluripotency (data not shown). No morphologicaldifferences could be observed between colonies grown in the testedculture systems and those grown on MEF in the presence of the yF10medium, correspondingly, morphological features remained unchanged on asingle-cell level, rendering cells small and round, exhibiting highnucleus-to-cytoplasm ratio, with a notable presence of one to threenucleoli and typical spacing between the cells (data not shown). Similarto cells grown on MEFs in the presence of a control medium (yF10 basicculture medium)), iPS cells or hESCs which were cultured on a Matrigel™(BD Biosceince) synthetic matrix in the presence of all of the testedmedia (HA74/1, HA75, HA76, HA77, HA78, HA40\4) were passaged routinelyevery five to seven days, at the same ratio of 1 to 2, 2 to 3, or 1 to3, indicating a similar population doubling time as iPS or hESCs grownon MEFs with the control medium. The iPS cells or the hESCs werepassaged at a same seeding efficiency of about 1 million cells per 10cm², with the same viability rate of over 90%. Using 15% serumreplacement (SR) and 10% DMSO, the iPS cells or the hESCs weresuccessfully frozen and thawed.

iPS cells or hESCs cultured on 2D culture systems in animal free mediumand supportive layer free system express markers of pluripotency—Severalsurface markers typical of primate undifferentiated ESCs and iPS cellswere examined using immunofluorescent staining [as described in Thomsonet al, 1995, 1996, 1998]. Cells cultured with the tested media for atleast 15 passages were found to be strongly positive to surface markersSSEA4, TRA-1-60. TRA-1-81 and Oct 4 (data not shown). As in otherprimate ESCs, staining with SSEA3 was weak and staining for SSEA1 wasnegative (data not shown).

iPS cells or hESCs cultured on 2D culture systems in animal free mediumand supportive layer free systems form EBs in vitro and teratomas invivo—The developmental potential of the cells after prolonged culture inthe tested conditions was examined in vitro by the formation of embryoidbodies (EBs). iPS or hESCs cells cultured in feeder layer-free culturesystems in the presence of the tested culture media (HA74/1, HA75, HA76,HA77, HA78, HA404) formed EBs similar to those created by ESCs grown onMEFs (data not shown). For example, the ability of iPS cells to form EBswas shown after 28 passages in the HA40/4 medium and 20 passages in theHA77 medium. Within these EBs, the iPS cells or the hESCs differentiatedinto cell types representative of the three embryonic germ layers[Itskovitz-eldor et al, 2000]. Following their injection to SCID Beigemice, the iPS cells or the hESCs cultured under the tested conditionsformed teratomas containing cell types representative of the threeembryonic germ layers (data not shown), thus demonstrating their fullpluripotency. For example, the ability of iPS cells to form teratomaswas shown after 31 passages in the mHA40/4 medium; after 24 passages inthe HA74/1 medium; and after 16 passages in the HA77 medium.

Example 2 Induced Pluripotent Stem Cells and Embryonic Stem Cells can beMaintained in an Undifferentiated and Pluripotent State when Cultured onXeno-Free Feeder-Layers in the Presence of Xeno-Free and Serum-FreeMedium

The experiments described hereinbelow were performed using iPS cells orhESCs which were cultured according to the methods, culturing conditionsand culture media described in the “General Materials and ExperimentalMethods” section above.

Experimental Results

iPS cells or hESCs cultured on 2D culture systems using xeno-free,serum-free medium and xeno-free feeder cell layers exhibitundifferentiated morphology and characteristics typical to iPS orhESCs—Several possible medium combinations (HA74/1, HA75, HA76, HA77.HA78. HA40\4) were tested for the ability to support xeno-free (devoidof any animal contaminant) cultures of iPS or hESCs using foreskinfibroblast as feeders cell layers. All tested media were found suitablefor supporting iPS or hESC cultures. Using the tested media underxeno-free conditions with foreskin fibroblasts as supportive layer, iPScells or hESCs were cultured continuously for at least 22 passages whilemaintaining their iPS or hESCs features including undifferentiatedproliferation (FIGS. 1A-C and data not shown), karyotype stability andpluripotency. No morphological differences could be observed betweencolonies grown in the tested culture systems and those grown on MEF inthe presence of the control yF10 medium, correspondingly, morphologicalfeatures remained unchanged on a single-cell level, rendering cellssmall and round, exhibiting high nucleus-to-cytoplasm ratio, with anotable presence of one to three nucleoli and typical spacing betweenthe cells (data not shown). Similar to cells grown on MEFs, iPS cells orhESCs cultured on foreskin fibroblast feeder cells in the presence ofall the tested culture media (HA74/1, HA75, HA76, HA77, HA78, HA404)were passaged routinely every five to seven days, at the same ratio of 1to 2, 2 to 3 or 1 to 3, indicating a similar population doubling time asiPS or hESCs grown on MEFs in the presence of a control yF10 medium. TheiPS cells or the hESCs were passaged at a same seeding efficiency ofabout 1 million cells per 10 cm², with the same viability rate of over90%. Using 15% serum replacement (SR) and 10% DMSO, the iPS cells or thehESCs were successfully frozen and thawed.

iPS cells or hESCs cultured on 2D culture systems in animal free mediumand xeno-free supportive layer express markers of pluripotency—Severalsurface markers typical of primate undifferentiated ESCs and iPS cellswere examined using immunofluorescent staining [as described in Thomsonet al, 1995, 1996, 1998]. Cells cultured with the tested media for atleast 15 passages were found to be strongly positive to surface markersSSEA4, TRA-1-60. TRA-1-81 and Oct 4 (FIGS. 2A-C). As in other primateESCs, staining with SSEA3 was weak and staining for SSEA1 was negative(data not shown).

iPS cells or hESCs cultured on 2D culture systems in animal free mediumand xeno-free feeder layers form EBs in vitro and teratomas in vivo—Thedevelopmental potential of the cells after prolonged culture in thetested conditions was examined in vitro by the formation of embryoidbodies (EBs). iPS cells or hESCs cultured in xeno-free feeder celllayers (foreskin fibroblasts) in the presence of the tested culturemedia (HA74/1. HA75. HA76, HA77. HA78, HA40\4) formed EBs similar tothose created by ESCs grown on MEFs in the presence of the yF10 controlmedium (data not shown). Within these EBs, the iPS cells or hESCsdifferentiated into cell types representative of the three embryonicgerm layers [Itskovitz-Eldor et al, 2000]. Following their injection toSCID Beige mice, the iPS cells or hESCs cultured under the testedconditions form teratomas containing cell types representative of thethree embryonic germ layers (data not shown), thus demonstrating theirfull pluripotency.

Example 3 Derivation of an Embryonic Stem Cell Line on the Xeno-FreeCulture Medium of the Invention

After digestion of the zona pellucida by Tyrode's acidic solution(Sigma, St Louis, Mo. USA), whole blastocysts were placed on mitoticallyinactivated human foreskin fibroblasts (HFF) in the presence of theHA40/4 medium, except that the medium did not contain sodiumbicarbonate. Initially, the cells were passage mechanically by usinginsulin syringes (BD plastipak. Cat. No. 300013) and after 4 passagesthe cells were passaged every four to six days using 1 mg/ml type IVcollagenase (Gibco Invitrogen corporation products, San Diego, Calif.USA). The resulting human ESC line was designated “WC1”.

Example 4 Induced Pluripotent Stem Cells and Embryonic Stem Cells can beMaintained in an Undifferentiated and Pluripotent State in StaticSuspension Cultures

Culture of iPS cells in suspension holds significant advantages overconventional cultures, particularly when aiming to obtain large amountsof cells for cell and tissue transplantation.

The experiments described hereinbelow were performed using iPS cells orhESCs which were cultured according to the methods, culturing conditionsand culture media described in the “General Materials and ExperimentalMethods” section above.

Experimental Results

iPS cells can be maintained in an undifferentiated state in suspensioncultures—The iPS cells (the J1.2-3 and iF4 cell lines) which were grownwith MEF or in feeder layer-free conditions [Amit et al. 2004], wereplaced in suspension cultures. After 24 hours in suspension culture withthe tested culture medium CM100F, CM100Fp, yFL3 (which comprises 4 ng/mlor 10 ng/ml bFGF and supplemented with 3000 units/ml LIF), or yF100, theiPS cells created spheroid clumps or disc-like structures which weremaintained for at least 20 passages (FIGS. 3A-C and data not shown).Histological examination of the iPS that were cultured in suspension forat least 10 passages revealed a homogenous population of small cellswith large nuclei. The spheroids grew and were split mechanically every5-7 days while maintaining their morphology, allowing expansion of thesuspension cultures. Alternatively, by using trypsin-EDTA and ROCKinhibitor treatment, suspended cells could be dissociated into singlecells and still formed spheroids of the same morphology and features,thus allowing efficient cell expansion. Some cultures were carried outfor over 50 passages (a year of continuous culture). The two differentiPS cell lines. 31.2-3 and iF4, which were cultured in suspension asdescribed herein with the tested culture media, showed similar behaviorand spheroid morphology and histology.

The yF100 medium (the yF10 basic culture medium which includes 100 ng/mlbFGF instead of 10 ng/ml), the CM100Fp and the yFL3 (the yF10 basicculture medium including 4 ng/ml bFGF instead of 10 ng/ml andsupplemented with 3000 units/ml LIF) were found to support the growth ofhuman ESCs in suspension culture in a proliferative, undifferentiatedand pluripotent state.

iPS cells which were cultured in suspension and were re-cultured on 2-Dculture systems maintain typical iPS cell colony morphology—After atleast 10 passages in suspension, when returned to 2D culture with MEFsor fibronectin surface, all of the spheroid clumps adhered to the MEFsor fibronectin surface and after 24-48 hours demonstrated typical iPScells colony morphology, exhibiting high nucleus-to-cytoplasm ratio witha notable presence of one to three nucleoli and with typical spacingbetween the cells (FIG. 4).

iPS cells maintain their undifferentiated stem cell phenotype whilebeing cultured in suspension cultures (3D cultures)—Several surfacemarkers typical of primate undifferentiated ESCs and iPS cells wereexamined using immunofluorescent staining [as described in Thomson etal, 1998; Bhattacharya, et a. 2004; Kristensen et al. 2005]. Human iPScells which were cultured in suspension with the tested culture mediafor at least 30 passages were found to be strongly positive for SSEA4,TRA-1-60 and TRA-1-81 and Oct 4 (FIGS. 5A-C). As with other primate ESCs[Thomson et al., 1995 and 1996] and with ESCs cultured with MEFs,staining with SSEA3 was weak and negative for SSEA1 data not shown).Staining for stem cell markers remained high when cells cultured insuspension were returned to 2D cultures with MEFs (data not shown).RT-PCR analyses showed that, similarly to cells cultured with MEFs, iPScells cultured in suspension for at least 10 passages expressed geneticmarkers of pluripotency [King et al, 2006] including Oct 4. Nanog. Sox2.Rex1, and FGF4 (data not shown). No significant difference in geneexpression of Oct 4, Nanog, Sox2, Rex1, and FGF4 was detected betweeniPS cells cultured in suspension as compared with iPS cells cultured onMEF, nor with iPS cells that were re-cultured with MEFs after continuousculture in suspension, similar to hESCs under the same conditions.

iPS cells which are cultured in suspension maintain normalkaryotype—Karyotype analysis by Giemsa banding was carried out on cellsafter 30 passages in suspension, and the cells were found to exhibitnormal 46XY karyotype (data not shown). Thus, the karyotype of thesuspension cell culture remained stable.

iPS cells or hESCs which are cultured in suspension maintain theirpluripotency in vitro—Following prolonged expansion in suspensioncultures with the tested culture media. iPS cells or hESCs preservedtheir pluripotent differentiation ability as was shown by the in vitroformation of EBs. When hESCs or iPS cells which were cultured insuspension for over 20 passages were transferred to serum-containingmedium without the addition of the growth factors, formation of cysticEBs was observed after 7-10 days, similarly to cavitated EBs formed fromhESCs following 10 days in culture [Itskovitz et al, 2000], and cysticEBs after 14-20 days. Within the EBs formed from the iPS cells or hESCs,there were cell types representative of the three embryonic germ layerstypical of iPS cells differentiation (data not shown).

For example, the ability of iPS cells to form EBs was shown after 22passages in the presence of the CM100p medium in a suspension culture;the ability to form EBs was shown after 23 passages in the presence ofthe yF100 medium in a suspension culture; the ability to form EBs wasshown after 8 passages in the presence of the yFL3 medium in asuspension culture.

iPS cells which are cultured in suspension maintain their pluripotencyin vivo—Pluripotency of the suspension iPS cells was furtherdemonstrated in vivo by teratoma formation. Cells cultured in suspensionfor at least 20 passages were injected into SCID Beige mice, and 10weeks later tumors were formed (data not shown). Within these teratomas,tissues representative of the three germ layers were observed.

For example, the ability of iPS cells to form teratomas was shown after20 passages in the CM100 in a suspension culture; and the ability toform teratomas was shown after 10 passages in the yFL3 in a suspensionculture.

Example 5 Induced Pluripotent Stem Cells and Embryonic Stem Cells can beMaintained in an Undifferentiated and Pluripotent State in DynamicSuspension Cultures

The experiments described hereinbelow were performed using iPS cells orhESCs which were cultured according to the methods, culturing conditionsand culture media described in the “General Materials and ExperimentalMethods” section above.

Experimental Results

iPS cells which are cultured in shaking suspension cultures maintaintheir undifferentiated state—iPS cells from line J1.2-3 or hESCs werecultured in suspension in spinner flasks for at least one month usingthe tested culture media. An examination after one month showed that themorphological characteristics of the spheroid clumps formed by the cellsremained similar to those observed when iPS cells are culturedstatically in Petri dishes (data not shown). In addition, the iPS cellsstrongly expressed markers of undifferentiated hESCs such as Oct-4,TRA-1-81, TRA-1-60 and SSEA4 (FIGS. 6A-D). When re-cultured on MEFs, theiPS cells in the clumps re-attached, forming again typical colonies ofiPS cells (data not shown). The karyotype of the cells cultured for onemonth in the spinner flask was found to be normal (data not shown).

iPS cells which are cultured in dynamic suspension cultures maintainnormal karyotype—IPS cells or hESCs which were cultured for 30 passagesin static suspension cultures (in the presence of the tested culturemedia) and then for 3 passages in dynamic (spinner) suspension (in thepresence of the tested culture media) were found to exhibit normal 46XYkaryotype. Thus, the karyotype of the suspension iPS cell cultureremained stable.

iPS cells of hESCs which are cultured in dynamic suspension maintaintheir pluripotency in vitro—The developmental potential of the iPS cellsor hESCs that were cultured in dynamic suspension cultures was examinedin vitro by the formation of EBs, hESCs or iPS were cultured in staticsuspension for over 20 passages, then on dynamic suspension for at leastadditional 10 passages, and then were transferred to serum-containingmedium without the addition of the growth factors, and the formation ofcystic EBs was observed after 7-10 days, similarly to cavitated EBSformed from hESCs following 10 days in culture [Itskovitz et al. 2000],and cystic EBs after 14-20 days. Within the EBs formed from hESCs or iPScells there were cell types representative of the three embryonic germlayers typical of iPS cells differentiation (data not shown).

iPS cells or hESCs which are cultured in dynamic suspension maintaintheir pluripotency in vivo—Pluripotency of iPS cells or hESCs culturedin dynamic suspension demonstrated in vivo by teratoma formation. Cellswere cultured in static suspension for at least 20 passages and then indynamic suspension for additional 10 passages and then were injectedinto SCID Beige mice. Following 10 weeks of injection into the micetumors were formed. Within these teratomas, tissues representative ofthe three germ layers were observed (data not shown).

This study presents a novel approach for culturing undifferentiated iPScells or human ESCs using either defined 2D culture system or suspensioncultures. The present inventors demonstrate that under these conditionstwo iPS cell lines, one derived from adult fibroblasts and one derivedfrom foreskin fibroblast could be grown and expanded through manypassages while maintaining their features including pluripotency andstable karyotypes. When iPS cells are transferred to suspension in thepresence of a differentiating medium (e.g., DMEM/F12 supplemented with10% fetal bovine serum (FBS). 10% KNOCKOUT™ serum replacement. 2 mML-glutamine, 0.1 mM β-mercaptoethanol, and 1% non-essential amino acidstock), they spontaneously form embryoid bodies (EBs). On the otherhand, using the tested culture systems (e.g., in the presence of theCM100F, CM100Fp, yF100 or yFL3 culture media) iPS cells spontaneouslyform spheroids consisting undifferentiated cells.

This is the first description of a method for continuous expansion ofundifferentiated iPS in 3D suspension and shaking cultures, which couldbe adequately applied for large-scale cell production.

The inventors present for the first time a suspension culture system forexpansion of undifferentiated iPS, based on serum free medium anddefined growth factors. This suspension culture system utilizes eitherPetri dishes, shaking Erlenmeyer, or spinner flasks. Two iPS cell linesfrom adult skin and newborn foreskin fibroblast were cultured accordingto the novel method of the invention as small spheroids which maintainall typical ESC/iPS cells features following prolonged culture of over25 passages (86 doublings), including stable karyotype and pluripotency.These results demonstrate that culturing iPS cells in a defined mediumwithout feeder layer using 3D culture is possible.

In addition, when applied onto a dynamic system for one month, thenumber of cell clumps of both hESCs and human iPS cells increased infolds while maintaining the cells unique characteristics. These resultsrender the proposed suspension system suitable for both the routineculture of iPS cells or hESCs in 3D and for mass production of iPS cellsand hESCs for therapeutic ends.

The teachings of the invention present scalable, reproducible andcontrolled culture systems. These results present a significant progresstowards the desired end goal of obtaining a facilitator method forlarge-scale culture of undifferentiated iPS cells and hESCs needed forboth clinical and industrial uses.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

It is the intent of the applicant(s) that all publications, patents andpatent applications referred to in this specification are to beincorporated in their entirety by reference into the specification, asif each individual publication, patent or patent application wasspecifically and individually noted when referenced that it is to beincorporated herein by reference. In addition, citation oridentification of any reference in this application shall not beconstrued as an admission that such reference is available as prior artto the present invention. To the extent that section headings are used,they should not be construed as necessarily limiting. In addition, anypriority document(s) of this application is/are hereby incorporatedherein by reference in its/their entirety.

REFERENCES Additional References are Cited in Text

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What is claimed is:
 1. A culture medium comprising an IL6RIL6 chimera ata concentration range of about 50-200 picogram per milliliter (μg/ml),wherein the culture medium is serum-free and capable of maintainingpluripotent stem cells in an undifferentiated state in the absence offeeder cell support.
 2. The culture medium of claim 1, wherein saidculture medium further comprises serum replacement.
 3. The culturemedium of claim 1, wherein said concentration of said IL6RIL6 chimera isbetween about 90 μg/ml to about 120 μg/ml.
 4. The culture medium ofclaim 1, wherein said concentration of said IL6RIL6 chimera is about 100μg/ml.
 5. The culture medium of claim 1, wherein said culture medium iscapable of maintaining and expanding said pluripotent stem cells in anundifferentiated state for at least 5 passages.
 6. The culture medium ofclaim 1, wherein said culture medium further comprises basic fibroblastgrowth factor (bFGF).
 7. A cell culture comprising pluripotent stemcells and the culture medium of claim
 1. 8. The cell culture of claim 7,wherein the cell culture is feeder cells free.
 9. The cell culture ofclaim 7, wherein said pluripotent stem cells are embryonic stem cells.10. The cell culture of claim 7, wherein said pluripotent stem cells areinduced pluripotent stem (iPS) cells.
 11. The cell culture of claim 7,wherein said pluripotent stem cells are human pluripotent stem cells.12. The cell culture of claim 7, being a suspension culture.
 13. Amethod of deriving an embryonic stem cell line, comprising (a) obtainingan embryonic stem cell from a pre-implantation stage blastocyst,post-implantation stage blastocyst and/or a genital tissue of a fetus;and (b) culturing said embryonic stem cell in the culture medium ofclaim 1; thereby deriving the embryonic stem cell line.
 14. A method ofderiving an induced pluripotent stem cell line, comprising (a) inducinga somatic cell to a pluripotent stem cell; and (b) culturing saidpluripotent stem cell in the culture medium of claim 1; thereby derivingthe induced pluripotent stem cell line.
 15. A method of expanding andmaintaining pluripotent stem cells in an undifferentiated state, themethod comprising culturing the pluripotent stem cells in the culturemedium of claim 1, thereby expanding and maintaining the pluripotentstem cells in the undifferentiated state.
 16. A method of generatinglineage-specific cells from pluripotent stem cells, the methodcomprising: (a) culturing the pluripotent stem cells in the culturemedium of claim 1, to thereby obtain expanded, undifferentiated stemcells; (b) subjecting said expanded, undifferentiated stem cells toculturing conditions suitable for differentiating and/or expandinglineage specific cells; thereby generating the lineage-specific cellsfrom the pluripotent stem cells.
 17. A method of generating embryoidbodies from pluripotent stem cells, the method comprising: (a) culturingthe pluripotent stem cells in the culture medium of claim 1, to therebyobtain expanded, undifferentiated pluripotent stem cells; and (b)subjecting said expanded, undifferentiated pluripotent stem cells toculturing conditions suitable for differentiating said stem cells toembryoid bodies; thereby generating the embryoid bodies from thepluripotent stem cells.
 18. A method of generating lineage-specificcells from pluripotent stem cells, the method comprising: (a) culturingthe pluripotent stem cells in the culture medium of claim 1, to therebyobtain expanded, undifferentiated pluripotent stem cells; (b) subjectingsaid expanded, undifferentiated pluripotent stem cells to culturingconditions suitable for differentiating said expanded, undifferentiatedstem cells to embryoid bodies; and (c) subjecting cells of said embryoidbodies to culturing conditions suitable for differentiating and/orexpanding lineage specific cells; thereby generating thelineage-specific cells from the pluripotent stem cells.
 19. The methodof claim 15, wherein said culturing is effected on a matrix.
 20. Themethod of claim 15, wherein said culturing is effected in a suspensionculture.
 21. The method of claim 15, wherein said culture medium isprotein carrier-free.